U.S. patent application number 11/230992 was filed with the patent office on 2006-04-06 for oligomeric compounds effecting drosha-mediated cleavage.
This patent application is currently assigned to ISIS Pharmaceuticals, Inc.. Invention is credited to Richard H. Griffey, Ravi Jain.
Application Number | 20060073505 11/230992 |
Document ID | / |
Family ID | 36090638 |
Filed Date | 2006-04-06 |
United States Patent
Application |
20060073505 |
Kind Code |
A1 |
Griffey; Richard H. ; et
al. |
April 6, 2006 |
Oligomeric compounds effecting drosha-mediated cleavage
Abstract
The present invention provides methods of promoting
Drosha-mediated cleavage of antisense oligomeric compounds and
compositions and compounds for carrying out the same.
Inventors: |
Griffey; Richard H.; (Vista,
CA) ; Jain; Ravi; (Carlsbad, CA) |
Correspondence
Address: |
COZEN O'CONNOR, P.C.
1900 MARKET STREET
PHILADELPHIA
PA
19103-3508
US
|
Assignee: |
ISIS Pharmaceuticals, Inc.
Carlsbad
CA
|
Family ID: |
36090638 |
Appl. No.: |
11/230992 |
Filed: |
September 20, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60612059 |
Sep 21, 2004 |
|
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|
Current U.S.
Class: |
435/6.12 ;
435/91.2; 536/23.1 |
Current CPC
Class: |
C12N 2310/341 20130101;
C12N 2310/315 20130101; C07H 21/02 20130101; C12Q 2521/307
20130101; C12N 2310/3525 20130101; C12Q 2525/131 20130101; C12N
2310/321 20130101; C12P 19/34 20130101; C12N 15/111 20130101; C12Q
1/68 20130101; C12N 2310/11 20130101; C12N 2330/30 20130101; C12Q
1/68 20130101; C12N 2310/321 20130101 |
Class at
Publication: |
435/006 ;
435/091.2; 536/023.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/02 20060101 C07H021/02; C12P 19/34 20060101
C12P019/34 |
Claims
1. A method of preparing an oligomeric compound that hybridizes to
a target mRNA comprising: incorporating a first region comprising
at least one nucleobase into the oligomeric compound that forms a
first 5' helical region with the target mRNA; incorporating a
second region comprising one or two mismatched nucleobases into the
oligomeric compound that forms a 5' destabilizing region with the
target mRNA; incorporating a third region comprising seven or eight
nucleobases into the oligomeric compound that forms a second 5'
helical region with the target mRNA; incorporating a fourth region
comprising two mismatched nucleobases into the oligomeric compound
that forms a cleavage signal region with the target mRNA;
incorporating a fifth region comprising four nucleobases into the
oligomeric compound that forms a cleavage site region with the
target mRNA; incorporating a sixth region comprising one or two
mismatched nucleobases into the oligomeric compound that forms a 3'
destabilizing region with the target mRNA; and incorporating a
seventh region comprising at least three nucleobases into the
oligomeric compound that forms a 3' helical region with the target
mRNA.
2. The method of claim 1 wherein the first region comprises at
least two nucleobases.
3. The method of claim 1 wherein the second region comprises one
nucleobase.
4. The method of claim 3 wherein the nucleobase forms a
pyrimidine/pyrimidine, A/C, or A/A mismatched base pair with the
target mRNA.
5. The method of claim 1 wherein the third region comprises seven
nucleobases.
6. The method of claim 1 wherein the third region does not comprise
a G/U base pair with the target mRNA.
7. The method of claim 1 wherein the fourth region comprises a
UU/UC, GG/AG, AG/AG, CA/CC, UG/CU, CU/CC, UA/GC, UC/UU, or
UU/G-mismatched base pair with the target mRNA.
8. The method of claim 1 wherein the sixth region comprises two
nucleobases.
9. The method of claim 8 wherein the sixth region comprises a GA/GG
mismatched base pair with the target mRNA.
10. The method of claim 1 wherein the sixth region comprises one
nucleobase.
11. The method of claim 10 wherein the sixth region comprises a C/C
mismatched base pair with the target mRNA.
12. The method of claim 1 wherein the fifth region comprises at
least one G/U base pair with the target mRNA.
13. The method of claim 1 wherein the oligomeric compound comprises
from about 18 to about 30 nucleobases.
14. The method of claim 1 wherein the oligomeric compound comprises
at least one nucleobase that comprises a
2'-O--CH.sub.2CH.sub.2OCH.sub.3 modification.
15. The method of claim 1 wherein the oligomeric compound is a
gapmer comprising three nucleobases phosphorothioate wings and a
phosphodiester gap, wherein each nucleobase within the wings
comprises a 2'-O--CH.sub.2CH.sub.2OCH.sub.3 modification.
16. A method of cleaving an mRNA target comprising contacting a
cell or tissue with an oligomeric compound that forms a duplex with
the mRNA target, wherein the duplex comprises: a first 5' helical
region comprising at least one base pair; a 5' destabilizing region
comprising one or two mismatched base pairs; a second 5' helical
region comprising seven or eight base pairs; a cleavage signal
region comprising two mismatched base pairs; a cleavage site region
comprising four base pairs; a 3' destabilizing region comprising
one or two mismatched base pairs; and a 3' helical region
comprising at least three base pairs.
17. The method of claim 16 wherein the first 5' helical region
comprises at least two base pairs.
18. The method of claim 16 wherein the 5' destabilizing region
comprises one mismatched base pair.
19. The method of claim 18 wherein the 5' destabilizing region
comprises a pyrimidine/pyrimidine, A/C, or A/A mismatched base
pair.
20. The method of claim 16 wherein the second 5' helical region
comprises seven base pairs.
21. The method of claim 16 wherein the second 5' helical region
does not comprise a G/U base pair.
22. The method of claim 16 wherein the cleavage signal region
comprises a UU/UC, GG/AG, AG/AG, CA/CC, UG/CU, CU/CC, UA/GC, UC/UU,
or UU/G-mismatched base pairs.
23. The method of claim 16 wherein the 3' destabilizing region
comprises two mismatched base pairs.
24. The method of claim 23 wherein the 3' destabilizing region
comprises a GA/GG mismatched base pairs.
25. The method of claim 16 wherein the 3' destabilizing region
comprises one mismatched base pair.
26. The method of claim 25 wherein the 3' destabilizing region
comprises a C/C mismatched base pair.
27. The method of claim 16 wherein the cleavage site region
comprises at least one G/U base pair.
28. The method of claim 16 wherein the oligomeric compound
comprises from about 18 to about 30 nucleobases.
29. The method of claim 16 wherein the oligomeric compound
comprises at least one nucleobase that comprises a
2'-O--CH.sub.2CH.sub.2OCH.sub.3 modification.
30. The method of claim 16 wherein the oligomeric compound is a
gapmer comprising three nucleobases phosphorothioate wings, wherein
each nucleobases within the wings comprises a
2'-O--CH.sub.2CH.sub.2OCH.sub.3 modification, and a phosphodiester
gap.
31. A composition comprising an oligomeric compound and an RNA
target wherein the oligomeric compound forms a duplex with the RNA
target, wherein the duplex comprises: a first 5' helical region
comprising at least one base pair; a 5' destabilizing region
comprising one or two mismatched base pairs; a second 5' helical
region comprising seven or eight base pairs; a cleavage signal
region comprising two mismatched base pairs; a cleavage site region
comprising four base pairs; a 3' destabilizing region comprising
one or two mismatched base pairs; and a 3' helical region
comprising at least three base pairs.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
application Ser. No. 60/612,059 filed Sep. 21, 2004, which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention is directed, in part, to methods of
promoting Drosha-mediated cleavage of antisense oligomeric
compounds and compositions and compounds for carrying out the
same.
BACKGROUND OF THE INVENTION
[0003] Conventional antisense oligomeric compounds work through the
action of endogenous RNAse H1, a ubiquitous enzyme that cleaves the
RNA strand of RNA-DNA duplexes. Human RNAse H has little or no
sequence dependence, but does require a minimum gap size of about 5
base pairs for substrate recognition.
[0004] RNAse III enzymes such as Dicer and Drosha form another
class of cellular RNAse activity. Dicer appears to prefer RNA
substrates with blunt ends or overhanging 3' bases associated with
a 5' phosphate residue. This terminus may be recognized by the PAZ
domain of Dicer to position the dual RNAse III domains prior to
substrate cleavage. Dicer is inhibited by long 5' or 3' single
stranded domains. Thus, Dicer is not well suited for
antisense-mediated cleavage.
[0005] Drosha is another cellular RNAse III enzyme first identified
by Wu et al. (J. Biol. Chem., 2000, 275, 36957-65) and McManus et
al. (RNA, 2002, 8, 842-850) and is involved in processing long
primary RNA transcripts (pri-miRNAs) from approximately 70 to 450
nucleotides in length into pre-miRNAs (from about 50 to about 80
nucleotides in length) which are exported from the nucleus to
encounter the human Dicer enzyme which then processes pre-miRNAs
into miRNAs. In cells, Drosha has been shown to cleave RNA Pol II
and Pol III transcripts associated with endogenous genes or
transfected expression vectors (Lee et al., Nature, 2003, 425,
415-419). It is believed that, in processing the pri-miRNA into the
pre-miRNA, the Drosha enzyme cuts the pri-miRNA at the base of the
mature miRNA, leaving a 2-nt 3'overhang (Lee et al., Nature, 2003,
425, 415-419). The 3' two-nucleotide overhang structure, a
signature of RNaseIII enzymatic cleavage, has been identified as a
critical specificity determinant in targeting and maintaining small
RNAs in the RNA interference pathway (Murchison et al., Curr. Opin.
Cell Biol., 2004, 16, 223-9).
[0006] The present invention is directed to harnessing Drosha to
effect Drosha-mediated cleavage of conventional antisense
oligomeric compounds.
SUMMARY OF THE INVENTION
[0007] The present invention provides methods of preparing an
oligomeric compound capable of undergoing Drosha-mediated cleavage.
In some embodiments, a Drosha-mediated cleavage recognition element
is incorporated in the oligomeric compound. In other embodiments, a
Drosha-mediated cleavage recognition element is identified in a
pri-miRNA and subsequently incorporated in the oligomeric
compound.
[0008] The present invention also provides methods of preparing an
oligomeric compound capable of undergoing Drosha-mediated cleavage.
The oligomeric compound is prepared such that it incorporates: a
first region comprising at least one nucleobase that forms a first
5' helical region with a target mRNA; a second region comprising
one or two mismatched nucleobases that forms a 5' destabilizing
region with the target mRNA; a third region comprising seven or
eight nucleobases that forms a second 5' helical region with the
target mRNA; a fourth region comprising two mismatched nucleobases
that forms a cleavage signal region with the target mRNA; a fifth
region comprising four nucleobases that forms a cleavage site
region with the target mRNA; a sixth region comprising one or two
mismatched nucleobases that forms a 3' destabilizing region with
the target mRNA; and a seventh region comprising at least three
nucleobases that forms a 3' helical region with the target mRNA. In
some embodiments, the first region comprises at least two
nucleobases. In some embodiments, the second region comprises one
nucleobases such as one that forms a pyrimidine/pyrimidine, A/C, or
A/A mismatched base pair with the target mRNA. In some embodiments,
the third region comprises seven nucleobases. In some embodiments,
the third region does not comprise a G/U base pair with the target
mRNA. In some embodiments, the fourth region comprises a UU/UC,
GG/AG, AG/AG, CA/CC, UG/CU, CU/CC, UA/GC, UC/UU, or UU/G-mismatched
base pair with the target mRNA. In some embodiments, the sixth
region comprises two nucleobases. In some embodiments, the sixth
region comprises a GA/GG mismatched base pair with the target mRNA.
In other embodiments, the sixth region comprises one nucleobases,
such as a C/C mismatched base pair with the target mRNA. In some
embodiments, the fifth region comprises at least one G/U base pair
with the target mRNA. In some embodiments, the oligomeric compound
comprises from about 13 to about 80 nucleobases, from about 13 to
about 50 nucleobases, from about 18 to about 30 nucleobases, from
about 19 to about 25 nucleobases, or from about 19 to about 22
nucleobases. In some embodiments, the oligomeric compound comprises
at least one nucleobase that comprises a
2'-O--CH.sub.2CH.sub.2OCH.sub.3 modification. In some embodiments,
the oligomeric compound is a gapmer comprising three nucleobases
phosphorothioate wings and a phosphodiester gap, wherein each
nucleobase within the wings comprises a
2'-O--CH.sub.2CH.sub.2OCH.sub.3 modification.
[0009] The present invention also provides methods of cleaving an
mRNA target comprising contacting a cell or tissue or animal with
an oligomeric compound that forms a duplex with the mRNA target. In
some embodiments, the duplex comprises: a first 5' helical region
comprising at least one base pair; a 5' destabilizing region
comprising one or two mismatched base pairs; a second 5' helical
region comprising seven or eight base pairs; a cleavage signal
region comprising two mismatched base pairs; a cleavage site region
comprising four base pairs; a 3' destabilizing region comprising
one or two mismatched base pairs; and a 3' helical region
comprising at least three base pairs. In some embodiments, the
first 5' helical region comprises at least two base pairs. In some
embodiments, the 5' destabilizing region comprises one mismatched
base pair. In some embodiments, the 5' destabilizing region
comprises a pyrimidine/pyrimidine, A/C, or A/A mismatched base
pair. In some embodiments, the second 5' helical region comprises
seven base pairs. In some embodiments, the second 5' helical region
does not comprise a G/U base pair. In some embodiments, the
cleavage signal region comprises a UU/UC, GG/AG, AG/AG, CA/CC,
UG/CU, CU/CC, UA/GC, UC/UU, or UU/G-mismatched base pairs. In some
embodiments, the 3' destabilizing region comprises two mismatched
base pairs. In some embodiments, the 3' destabilizing region
comprises a GA/GG mismatched base pairs. In some embodiments, the
3' destabilizing region comprises one mismatched base pair. In some
embodiments, the 3' destabilizing region comprises a C/C mismatched
base pair. In some embodiments, the cleavage site region comprises
at least one G/U base pair. In some embodiments, the oligomeric
compound comprises from about 13 to about 80 nucleobases, from
about 13 to about 50 nucleobases, from about 18 to about 30
nucleobases, from about 19 to about 25 nucleobases, or from about
19 to about 22 nucleobases. In some embodiments, the oligomeric
compound comprises at least one nucleobase that comprises a
2'-O--CH.sub.2CH.sub.2OCH.sub.3 modification. In some embodiments,
the oligomeric compound is a gapmer comprising three nucleobases
phosphorothioate wings, wherein each nucleobases within the wings
comprises a 2'-O--CH.sub.2CH.sub.2OCH.sub.3 modification, and a
phosphodiester gap.
[0010] The present invention also provides compositions comprising
an oligomeric compound and an RNA target, wherein the oligomeric
compound forms a duplex with the RNA target. In some embodiments,
the duplex comprises: a first 5' helical region comprising at least
one base pair; a 5' destabilizing region comprising one or two
mismatched base pairs; a second 5' helical region comprising seven
or eight base pairs; a cleavage signal region comprising two
mismatched base pairs; a cleavage site region comprising four base
pairs; a 3' destabilizing region comprising one or two mismatched
base pairs; and a 3' helical region comprising at least three base
pairs.
[0011] The present invention also provides oligomeric compounds
that when duplexed to an RNA target comprise: a first 5' helical
region comprising at least one base pair; a 5' destabilizing region
comprising one or two mismatched base pairs; a second 5' helical
region comprising seven or eight base pairs; a cleavage signal
region comprising two mismatched base pairs; a cleavage site region
comprising four base pairs; a 3' destabilizing region comprising
one or two mismatched base pairs; and a 3' helical region
comprising at least three base pairs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows a sequence alignment of 50 human pri-mir
sequences.
[0013] FIG. 2 shows a representative motif to search for in a
target mRNA.
[0014] FIG. 3 shows a representative motif to search for in a
target mRNA.
[0015] FIG. 4A shows representative effects of uniform PO-MOE in
HeLa cells with Drosha sequences at 150 nM for 16 hours.
[0016] FIG. 4B shows representative effects of 3-base MOE PS wings,
RNA PO gap in HeLa cells with Drosha sequences at 150 nM for 16
hours.
[0017] FIG. 4C shows representative effects of uniform RNA PS with
one PO at position 14 in HeLa cells with Drosha sequences at 150 nM
for 16 hours.
DESCRIPTION OF EMBODIMENTS
[0018] The present invention provides methods of preparing an
oligomeric compound capable of undergoing Drosha-mediated cleavage,
methods of cleaving an mRNA target by contacting a cell or tissue
with an oligomeric compound that forms a duplex with the mRNA
target, methods of cleaving an mRNA target in an animal by
contacting the animal with an oligomeric compound that forms a
duplex with the mRNA target, and compositions and compounds.
[0019] The present invention provides methods of preparing an
oligomeric compound capable of undergoing Drosha-mediated cleavage.
As used herein, "Drosha-mediated cleavage" means any cleavage of an
oligomeric compound in which Drosha participates. In some
embodiments, one or more Drosha-mediated cleavage recognition
elements are incorporated into an oligomeric compound, such that
when the oligomeric compound forms a duplex with an mRNA target,
the duplex is cleaved in a Drosha-mediated manner. As used herein,
a "Drosha-mediated cleavage recognition element" is any element
within a polynucleotide sequence that causes the polynucleotide to
be recognized by Drosha. The Drosha-mediated cleavage recognition
element can be a particular nucleobase in a particular location,
can be a particular base pairing between the oligomeric compound
and the mRNA target, or can be a structural component. In some
embodiments, a Drosha-mediated cleavage recognition element is
first identified in a pri-miRNA and subsequently incorporated in
the oligomeric compound.
[0020] In some embodiments, an oligomeric compound capable of
undergoing Drosha-mediated cleavage is prepared by routine
procedures well known to the skilled artisan. The oligomeric
compound, however, is designed to contain one or more
Drosha-mediated recognition elements. For example, an oligomeric
compound capable of undergoing Drosha-mediated cleavage can be
designed to have one or more, or any combination thereof, of the
following Drosha-mediated recognition elements. The Drosha-mediated
recognition elements are set forth below as regions of the
oligomeric compound that interact and form a duplex with the target
mRNA. A representative oligomeric compound can, thus have the
following formula: 3' helical region--3' destabilizing
region--cleavage site region--cleavage signal region--second 5'
helical region--5' destabilizing region--first 5' helical region.
Other oligomeric compounds can have various region(s) omitted.
[0021] One Drosha-mediated recognition element that can be
incorporated into an oligomeric compound is a first region that
comprises at least one nucleobase that can form a first 5' helical
region with a target mRNA. In some embodiments, the 5' helical
region comprises at least two nucleobases. In some embodiments, a
5' helical region that comprises 2 or more nucleobases can also
comprise bulges or mismatched base pairs.
[0022] Another Drosha-mediated recognition element that can be
incorporated into an oligomeric compound is a second region that
comprises one or two mismatched nucleobases that can form a 5'
destabilizing region with the target mRNA. In some embodiments, the
5' destabilizing region comprises one nucleobases, such as a
nucleobases that can form a pyrimidine/pyrimidine, A/C, or A/A
mismatched base pair with the target mRNA. The 5' destabilizing
region can comprise more than two mismatched base pairs as long as
it retains the ability to form a stable duplex with the target. In
some embodiments, the 5' destabilizing region can comprise
nucleobases that can undergo normal Watson-Crick base pairing (e.g,
A-T, C-G; U-A) but which comprise a chemical modification that
renders it incapable of forming such a Watson-Crick base pairing
(e.g., 3-methyluridine, 4-thiouridine, 6-thioguanosine, N-1-methyl
guanosine, N,N-dimethylaminoguanosine, N,N-dimethylaminoadenosine,
2-thiomethyladenosine, and the like). Additional mismatched base
pairs can be tolerated by the enzyme, although they may have a
lower natural frequency among known Drosha substrates.
[0023] Another Drosha-mediated recognition element that can be
incorporated into an oligomeric compound is a third region that
comprises seven or eight nucleobases that can form a second 5'
helical region with the target mRNA. In some embodiments, the
second 5' helical region comprises seven nucleobases. In some
embodiments, the second 5' helical region does not comprise a G/U
base pair with the target mRNA. The second 5' helical region can be
more than seven nucleobases, and can comprise as many as fifteen
nucleobases.
[0024] Another Drosha-mediated recognition element that can be
incorporated into an oligomeric compound is a fourth region that
comprises two mismatched nucleobases that can form a cleavage
signal region with the target mRNA. In some embodiments, the
cleavage signal region comprises a UU/UC, GG/AG, AG/AG, CA/CC,
UG/CU, CU/CC, UA/GC, UC/UU, or UU/G-mismatched base pair with the
target mRNA. The cleavage signal region can comprise more than two
mismatched base pairs as long as it retains the ability to form a
stable duplex with the target. In some embodiments, the cleavage
signal region can comprise nucleobases that can undergo normal
Watson-Crick base pairing (e.g, A-T, C-G; U-A) but which comprise a
chemical modification that renders it incapable of forming such a
Watson-Crick base pairing (e.g., 3-methyluridine, 4-thiouridine,
6-thioguanosine, N-1-methylguanosine, N,N-dimethylaminoguanosine,
N,N-dimethylaminoadenosine, 2-thiomethyladenosine, and the like).
Additional mismatched base pairs can be tolerated by the enzyme,
although they may have a lower natural frequency among known Drosha
substrates.
[0025] Another Drosha-mediated recognition element that can be
incorporated into an oligomeric compound is a fifth region that
comprises two to four nucleobases that can form a cleavage site
region with the target mRNA. In some embodiments, the cleavage site
region comprises at least one G/U base pair with the target
mRNA.
[0026] Another Drosha-mediated recognition element that can be
incorporated into an oligomeric compound is a sixth region that
comprises one or two mismatched nucleobases that can form a 3'
destabilizing region with the target mRNA. In some embodiments, the
3' destabilizing region comprises two nucleobases, such as two
nucleobases that form a GA/GG mismatched base pair with the target
mRNA. In other embodiments, the 3' destabilizing region comprises
one nucleobases, such as one nucleobase that forms a C/C mismatched
base pair with the target mRNA. The 3' destabilizing region can
comprise more than two mismatched base pairs as long as it retains
the ability to form a stable duplex with the target. In some
embodiments, the 3' destabilizing region can comprise nucleobases
that can undergo normal Watson-Crick base pairing (e.g, A-T, C-G;
U-A) but which comprise a chemical modification that renders it
incapable of forming such a Watson-Crick base pairing (e.g.,
3-methyluridine, 4-thiouridine, 6-thioguanosine,
N-1-methylguanosine, N,N-dimethylaminoguanosine,
N,N-dimethylaminoadenosine, 2-thiomethyladenosine, and the like).
Additional mismatched base pairs can be tolerated by the enzyme,
although they may have a lower natural frequency among known Drosha
substrates.
[0027] Another Drosha-mediated recognition element that can be
incorporated into an oligomeric compound is a seventh region that
comprises at least three nucleobases that can form a 3' helical
region with the target mRNA.
[0028] Other Drosha-mediated recognition elements known to those
skilled in the art can also be incorporated into an oligomeric
compound to promote Drosha-mediated cleavage of the oligomeric
compound. For example, a stem-loop on the 5' end of the antisense
strand may function as a Drosha-mediated recognition element.
[0029] The present invention also provides methods of cleaving an
mRNA target comprising contacting an animal, cell, or tissue with
any of the oligomeric compounds described herein that can form a
duplex with the mRNA target. Such oligomeric compounds can be used,
for example, to treat conditions or diseases that are linked to a
particular gene or mRNA. The present invention also provides use of
any of the oligomeric compounds of the present invention that can
undergo Drosha-mediated cleavage in the formation of a medicament
for treating a condition or disease linked to a particular gene or
mRNA.
[0030] As used herein, "mRNA target" means any mRNA capable of
being targeted by an olifomeric compound. These targets can be
pre-mRNAs or mRNAs; single- or double-stranded, or single-stranded
with partial double-stranded character; may occur naturally within
introns or exons of messenger RNAs (mRNAs); and can be endogenously
transcribed or exogenously produced.
[0031] In the context of the present invention, the term
"oligomeric compound(s)" refers to a polymer or oligomer comprising
a plurality of monomeric units. In the context of this invention,
the term "oligomeric compound" refers to an oligomer or polymer of
ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics,
chimeras, analogs and homologs thereof. This term includes
oligonucleotides composed of naturally occurring nucleobases,
sugars and covalent internucleoside (backbone) linkages as well as
oligonucleotides having non-naturally occurring portions which
function similarly. Such modified or substituted oligonucleotides
are often preferred over native forms because of desirable
properties such as, for example, enhanced cellular uptake, enhanced
affinity for a target nucleic acid and increased stability in the
presence of nucleases. The term "oligomeric compound" includes, but
is not limited to, compounds comprising oligonucleotides,
oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics
and combinations of these. Oligomeric compounds also include, but
are not limited to, antisense oligomeric compounds, antisense
oligonucleotides, siRNAs, alternate splicers, primers, probes and
other compounds that hybridize to at least a portion of the target
nucleic acid. Oligomeric compounds are routinely prepared linearly
but can be joined or otherwise prepared to be circular and may also
include branching. Separate oligomeric compounds can hybridize to
form double stranded compounds that can be blunt-ended or may
include overhangs on one or both termini. In general, an oligomeric
compound comprises a backbone of linked monomeric subunits where
each linked monomeric subunit is directly or indirectly attached to
a heterocyclic base moiety. The linkages joining the monomeric
subunits, the sugar moieties or sugar surrogates and the
heterocyclic base moieties can be independently modified giving
rise to a plurality of motifs for the resulting oligomeric
compounds including hemimers, gapmers and chimeras.
[0032] As is known in the art, a nucleoside is a base-sugar
combination. The base portion of the nucleoside is normally a
heterocyclic base moiety. The two most common classes of such
heterocyclic bases are purines and pyrimidines. Nucleotides are
nucleosides that further include a phosphate group covalently
linked to the sugar portion of the nucleoside. For those
nucleosides that include a pentofuranosyl sugar, the phosphate
group can be linked to either the 2', 3' or 5' hydroxyl moiety of
the sugar. In forming oligonucleotides, the phosphate groups
covalently link adjacent nucleosides to one another to form a
linear polymeric compound. The respective ends of this linear
polymeric structure can be joined to form a circular structure by
hybridization or by formation of a covalent bond. In addition,
linear compounds may have internal nucleobase complementarity and
may therefore fold in a manner as to produce a fully or partially
double-stranded structure. Within the oligonucleotide structure,
the phosphate groups are commonly referred to as forming the
internucleoside linkages of the oligonucleotide. The normal
internucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester
linkage.
[0033] In the context of this invention, the term "oligonucleotide"
refers generally to an oligomer or polymer of ribonucleic acid
(RNA) or deoxyribonucleic acid (DNA). This term includes
oligonucleotides composed of naturally occurring nucleobases,
sugars and covalent internucleoside linkages. The term
"oligonucleotide analog" refers to oligonucleotides that have one
or more non-naturally occurring portions which function in a
similar manner to oligonucleotides. Such non-naturally occurring
oligonucleotides are often selected over naturally occurring forms
because of desirable properties such as, for example, enhanced
cellular uptake, enhanced affinity for other oligonucleotides or
nucleic acid targets and increased stability in the presence of
nucleases.
[0034] In the context of this invention, the term "oligonucleoside"
refers to nucleosides that are joined by internucleoside linkages
that do not have phosphorus atoms. Internucleoside linkages of this
type include short chain alkyl, cycloalkyl, mixed heteroatom alkyl,
mixed heteroatom cycloalkyl, one or more short chain heteroatomic
and one or more short chain heterocyclic. These internucleoside
linkages include but are not limited to siloxane, sulfide,
sulfoxide, sulfone, acetyl, formacetyl, thioformacetyl, methylene
formacetyl, thioformacetyl, alkeneyl, sulfamate; methyleneimino,
methylenehydrazino, sulfonate, sulfonamide, amide and others having
mixed N, O, S and CH.sub.2 component parts. In addition to the
modifications described above, the nucleosides of the oligomeric
compounds of the invention can have a variety of other
modifications. Additional nucleosides amenable to the present
invention having altered base moieties and or altered sugar
moieties are disclosed in U.S. Pat. No. 3,687,808 and PCT
application PCT/US89/02323.
[0035] For nucleotides that are incorporated into oligonucleotides
of the invention, these nucleotides can have sugar portions that
correspond to naturally occurring sugars or modified sugars.
Representative modified sugars include carbocyclic or acyclic
sugars, sugars having substituent groups at one or more of their
2', 3' or 4' positions and sugars having substituents in place of
one or more hydrogen atoms of the sugar.
[0036] Altered base moieties or altered sugar moieties also include
other modifications consistent with the spirit of this invention.
Such oligomeric compounds are best described as being structurally
distinguishable from, yet functionally interchangeable with,
naturally occurring or synthetic unmodified oligonucleotides. All
such oligomeric compounds are comprehended by this invention so
long as they function effectively to mimic the structure or
function of a desired RNA or DNA oligonucleotide strand.
[0037] A class of representative base modifications include
tricyclic cytosine analog, termed "G clamp" (Lin et al., J. Am.
Chem. Soc., 1998, 120, 8531). This analog can form four hydrogen
bonds with a complementary guanine (G) by simultaneously
recognizing the Watson-Crick and Hoogsteen faces of the targeted G.
This G clamp modification when incorporated into phosphorothioate
oligomeric compounds, dramatically enhances potencies as measured
by target reduction in cell culture. The oligomeric compounds of
the invention also can include phenoxazine-substituted bases of the
type disclosed by Flanagan et al., Nat. Biotechnol., 1999, 17,
48-52.
[0038] The oligomeric compounds in accordance with the present
invention can comprise from about 13 to about 80 monomeric subunits
(i.e., from about 13 to about 80 linked nucleosides). One of
ordinary skill in the art will appreciate that the invention
embodies oligomeric compounds of 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, or 80 subunits in length, or any
range therewithin.
[0039] The oligomeric compounds in accordance with the present
invention can also comprise from about 13 to about 50 monomeric
subunits (i.e., from about 13 to about 50 linked nucleosides). One
of ordinary skill in the art will appreciate that the invention
embodies oligomeric compounds of 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 subunits in
length, or any range therewithin.
[0040] The oligomeric compounds in accordance with the present
invention can also comprise from about 18 to about 30 monomeric
subunits (i.e., from about 18 to about 30 linked nucleosides). One
of ordinary skill in the art will appreciate that the invention
embodies oligomeric compounds of 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, or 30 subunits in length, or any range
therewithin.
[0041] The oligomeric compounds in accordance with the present
invention can also comprise from about 19 to about 25 monomeric
subunits (i.e., from about 19 to about 25 linked nucleosides). One
of ordinary skill in the art will appreciate that the invention
embodies oligomeric compounds of 19, 20, 21, 22, 23, 24, or 25
subunits in length, or any range therewithin.
[0042] The oligomeric compounds in accordance with the present
invention can also comprise from about 19 to about 22 monomeric
subunits (i.e., from about 19 to about 22 linked nucleosides). One
of ordinary skill in the art will appreciate that the invention
embodies oligomeric compounds of 19, 20, 21, or 22 subunits in
length, or any range therewithin.
[0043] "Targeting" an oligomeric compound to a particular nucleic
acid molecule, in the context of this invention, can be a multistep
process. The process usually begins with the identification of a
target nucleic acid whose levels, expression or function is to be
modulated. This target nucleic acid may be, for example, a mRNA
transcribed from a cellular gene whose expression is associated
with a particular disorder or disease state, a small non-coding RNA
or its precursor, or a nucleic acid molecule from an infectious
agent.
[0044] The targeting process usually also includes determination of
at least one target region, segment, or site within the target
nucleic acid for the interaction to occur such that the desired
effect, e.g., modulation of levels, expression or function, will
result. Within the context of the present invention, the term
"region" is defined as a portion of the target nucleic acid having
at least one identifiable sequence, structure, function, or
characteristic. Within regions of target nucleic acids are
segments. "Segments" are defined as smaller or sub-portions of
regions within a target nucleic acid. "Sites," as used in the
present invention, are defined as specific positions within a
target nucleic acid. The terms region, segment, and site can also
be used to describe an oligomeric compound of the invention such as
for example a gapped oligomeric compound having three separate
segments.
[0045] Targets of the present invention include both coding and
non-coding nucleic acid sequences. For coding nucleic acid
sequences, the translation initiation codon is typically 5'-AUG (in
transcribed mRNA molecules; 5'-ATG in the corresponding DNA
molecule), the translation initiation codon is also referred to as
the "AUG codon," the "start codon" or the "AUG start codon." A
minority of genes have a translation initiation codon having the
RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and
5'-CUG have been shown to function in vivo. Thus, the terms
"translation initiation codon" and "start codon" can encompass many
codon sequences, even though the initiator amino acid in each
instance is typically methionine (in eukaryotes) or
formylmethionine (in prokaryotes). It is also known in the art that
eukaryotic and prokaryotic genes may have two or more alternative
start codons, any one of which may be preferentially utilized for
translation initiation in a particular cell type or tissue, or
under a particular set of conditions. In the context of the
invention, "start codon" and "translation initiation codon" refer
to the codon or codons that are used in vivo to initiate
translation of an mRNA transcribed from a gene encoding a nucleic
acid target, regardless of the sequence(s) of such codons. It is
also known in the art that a translation termination codon (or
"stop codon") of a gene may have one of three sequences, i.e.,
5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are
5'-TAA, 5'-TAG and 5'-TGA, respectively).
[0046] The terms "start codon region" and "translation initiation
codon region" refer to a portion of such an mRNA or gene that
encompasses from about 25 to about 50 contiguous nucleotides in
either direction (i.e., 5' or 3') from a translation initiation
codon. Similarly, the terms "stop codon region" and "translation
termination codon region" refer to a portion of such an mRNA or
gene that encompasses from about 25 to about 50 contiguous
nucleotides in either direction (i.e., 5' or 3') from a translation
termination codon. Consequently, the "start codon region" (or
"translation initiation codon region") and the "stop codon region"
(or "translation termination codon region") are all regions which
may be targeted effectively with the oligomeric compounds of the
present invention.
[0047] The open reading frame (ORF) or "coding region," which is
known in the art to refer to the region between the translation
initiation codon and the translation termination codon, is also a
region which may be targeted effectively. Within the context of the
present invention, a further suitable region is the intragenic
region encompassing the translation initiation or termination codon
of the open reading frame (ORF) of a gene.
[0048] Other target regions include the 5' untranslated region
(5'UTR), known in the art to refer to the portion of an mRNA in the
5' direction from the translation initiation codon, and thus
including nucleotides between the 5' cap site and the translation
initiation codon of an mRNA (or corresponding nucleotides on the
gene), and the 3' untranslated region (3'UTR), known in the art to
refer to the portion of an mRNA in the 3' direction from the
translation termination codon, and thus including nucleotides
between the translation termination codon and 3' end of an mRNA (or
corresponding nucleotides on the gene). The 5' cap site of an mRNA
comprises an N7-methylated guanosine residue joined to the 5'-most
residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap
region of an mRNA is considered to include the 5' cap structure
itself as well as the first 50 nucleotides adjacent to the cap
site. It is also suitable to target the 5' cap region.
[0049] Although some eukaryotic mRNA transcripts are directly
translated, many contain one or more regions, known as "introns,"
which are excised from a transcript before it is translated. The
remaining (and therefore translated) regions are known as "exons"
and are spliced together to form a continuous mRNA sequence.
Targeting splice sites, i.e., intron-exon junctions or exon-intron
junctions, may also be particularly useful in situations where
aberrant splicing is implicated in disease, or where an
overproduction of a particular splice product is implicated in
disease. Aberrant fusion junctions due to rearrangements or
deletions are also target sites. mRNA transcripts produced via the
process of splicing of two (or more) mRNAs from different gene
sources are known as "fusion transcripts." It is also known that
introns can be effectively targeted using oligomeric compounds
targeted to, precursor molecules for example, pre-mRNA.
[0050] It is also known in the art that alternative RNA transcripts
can be produced from the same genomic region of DNA. These
alternative transcripts are generally known as "variants." More
specifically, "pre-mRNA variants" are transcripts produced from the
same genomic DNA that differ from other transcripts produced from
the same genomic DNA in either their start or stop position and
contain both intronic and exonic sequences.
[0051] Upon excision of one or more exon or intron regions, or
portions thereof, during splicing, pre-mRNA variants produce
smaller "mRNA variants." Consequently, mRNA variants are processed
pre-mRNA variants and each unique pre-mRNA variant must always
produce a unique mRNA variant as a result of splicing. These mRNA
variants are also known as "alternative splice variants." If no
splicing of the pre-mRNA variant occurs then the pre-mRNA variant
is identical to the mRNA variant.
[0052] It is also known in the art that variants can be produced
through the use of alternative signals to start or stop
transcription and that pre-mRNAs and mRNAs can possess more that
one start codon or stop codon. Variants that originate from a
pre-mRNA or mRNA that use alternative start codons are known as
"alternative start variants" of that pre-mRNA or mRNA. Those
transcripts that use an alternative stop codon are known as
"alternative stop variants" of that pre-mRNA or mRNA. One specific
type of alternative stop variant is the "polyA variant" in which
the multiple transcripts produced result from the alternative
selection of one of the "polyA stop signals" by the transcription
machinery, thereby producing transcripts that terminate at unique
polyA sites. Within the context of the invention, the types of
variants described herein are also target nucleic acids.
[0053] Once one or more targets, target regions, segments or sites
have been identified, oligomeric compounds are designed to be
sufficiently complementary to the target, i.e., hybridize
sufficiently well and with sufficient specificity, to give the
desired effect. The desired effect may include, but is not limited
to modulation of the levels, expression or function of the
target.
[0054] The oligomeric compounds of the present invention can also
comprise one or more chemical modifications, such as modifications
of the sugar, nucleobase, or internucleoside linkage. In some
embodiments, the oligomeric compound comprises at least one
2'-O--CH.sub.2CH.sub.2OCH.sub.3 modification. In some embodiments,
the oligomeric compound is a gapmer comprising three nucleobases
phosphorothioate wings and a phosphodiester gap, wherein each
nucleobase within the wings comprises a
2'-O--CH.sub.2CH.sub.2OCH.sub.3 modification.
[0055] Oligomerization of modified and unmodified nucleosides is
performed according to literature procedures for DNA like compounds
(Protocols for Oligonucleotides and Analogs, Ed. Agrawal, 1993,
Humana Press) and/or RNA like compounds (Scaringe, Methods, 2001,
23, 206-217; Gait et al., Applications of Chemically synthesized
RNA in RNA:Protein Interactions, Ed. Smith, 1998, 1-36; and Gallo
et al., Tetrahedron, 2001, 57, 5707-5713) synthesis as appropriate.
In addition, specific protocols for the synthesis of oligomeric
compounds of the invention are illustrated in the examples
below.
[0056] RNA oligomers can be synthesized by methods disclosed herein
or purchased from various RNA synthesis companies such as for
example Dharmacon Research Inc., (Lafayette, Colo.).
[0057] Irrespective of the particular protocol used, the oligomeric
compounds used in accordance with this invention may be
conveniently and routinely made through the well-known technique of
solid phase synthesis. Equipment for such synthesis is sold by
several vendors including, for example, Applied Biosystems (Foster
City, Calif.). Any other means for such synthesis known in the art
may additionally or alternatively be employed.
[0058] Synthesis of Nucleoside Phosphoramidites: The following
compounds, including amidites and their intermediates were prepared
as described in U.S. Pat. No. 6,426,220 and published PCT WO
02/36743; 5'-O-Dimethoxytrityl-thymidine intermediate for 5-methyl
dC amidite, 5'-O-Dimethoxytrityl-2'-deoxy-5-methylcytidine
intermediate for 5-methyl-dC amidite,
5'-O-Dimethoxytrityl-2'-deoxy-N-4-benzoyl-5-methylcytidine
penultimate intermediate for 5-methyl dC amidite,
(5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-deoxy-N.sup.4-benzoyl-5-methylcy-
tidin-3'-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite
(5-methyl dC amidite), 2'-Fluorodeoxyadenosine,
2'-Fluorodeoxyguanosine, 2'-Fluorouridine, 2'-Fluorodeoxycytidine,
2'-O-(2-Methoxyethyl) modified amidites,
2'-O-(2-methoxyethyl)-5-methyluridine intermediate,
5'-O-DMT-2'-O-(2-methoxyethyl)-5-methyluridine penultimate
intermediate,
(5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-5-methyluridi-
n-3'-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T
amidite),
5'-O-Dimethoxytrityl-2'-O-(2-methoxyethyl)-5-methylcytidine
intermediate,
5'-O-dimethoxytrityl-2'-O-(2-methoxyethyl)-N.sup.4-benzoyl-5-methyl-cytid-
ine penultimate intermediate,
(5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.sup.4-benzo-
yl-5-methylcytidin-3'-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite
(MOE 5-Me-C amidite),
(5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.sup.6-benzo-
yladenosin-3'-O-yl)-2cyanoethyl-N,N-diisopropylphosphoramidite (MOE
A amdite),
(5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.su-
p.4-isobutyrylguanosin-3'-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidit-
e (MOE G amidite), 2'-O-(Aminooxyethyl) nucleoside amidites and
2'-O-(dimethylaminooxyethyl) nucleoside amidites,
2'-(Dimethylaminooxyethoxy) nucleoside amidites,
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-5-methyluridine,
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine,
2'-O-((2-phthalimidoxy)ethyl)-5'-t-butyldiphenylsilyl-5-methyluridine,
5'-O-tert-butyldiphenylsilyl-2'-O-((2-formadoximinooxy)ethyl)-5-methyluri-
dine, 5'-O-tert-Butyldiphenylsilyl-2'-O-(N,N
dimethylaminooxyethyl)-5-methyluridine,
2'-O-(dimethylaminooxyethyl)-5-methyluridine,
5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine,
5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-((2-cyanoe-
thyl)-N,N-diisopropylphosphoramidite), 2'-(Aminooxyethoxy)
nucleoside amidites,
N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(-
4,4'-dimethoxytrityl)guanosine-3'-((2-cyanoethyl)-N,N-diisopropylphosphora-
midite), 2'-dimethylaminoethoxyethoxy (2'-DMAEOE) nucleoside
amidites, 2'-O-(2(2-N,N-dimethylaminoethoxy)ethyl)-5-methyl
uridine,
5'-O-dimethoxytrityl-2'-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyl
uridine and
5'-O-Dimethoxytrityl-2'-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methylu-
ridine-3'-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.
[0059] Oligonucleotides: Unsubstituted and substituted
phosphodiester (P.dbd.O) oligonucleotides are synthesized on an
automated DNA synthesizer (Applied Biosystems model 394) using
standard phosphoramidite chemistry with oxidation by iodine.
[0060] Phosphorothioates (P.dbd.S) are synthesized similar to
phosphodiester oligonucleotides with the following exceptions:
thiation was effected by utilizing a 10% w/v solution of
3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the
oxidation of the phosphite linkages. The thiation reaction step
time was increased to 180 sec and preceded by the normal capping
step. After cleavage from the CPG column and deblocking in
concentrated ammonium hydroxide at 55.degree. C. (12-16 hr), the
oligonucleotides were recovered by precipitating with >3 volumes
of ethanol from a 1 M NH.sub.4OAc solution. Phosphinate
oligonucleotides are prepared as described in U.S. Pat. No.
5,508,270.
[0061] Alkyl phosphonate oligonucleotides are prepared as described
in U.S. Pat. No. 4,469,863.
[0062] 3'-Deoxy-3'-methylene phosphonate oligonucleotides are
prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050.
[0063] Phosphoramidite oligonucleotides are prepared as described
in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878.
[0064] Alkylphosphonothioate oligonucleotides are prepared as
described in published PCT applications WO 94/17093 and WO
94/02499.
[0065] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are
prepared as described in U.S. Pat. No. 5,476,925.
[0066] Phosphotriester oligonucleotides are prepared as described
in U.S. Pat. No. 5,023,243.
[0067] Borano phosphate oligonucleotides are prepared as described
in U.S. Pat. Nos. 5,130,302 and 5,177,198.
[0068] Oligonucleosides: Methylenemethylimino linked
oligonucleosides, also identified as MMI linked oligonucleosides,
methylenedimethylhydrazo linked oligonucleosides, also identified
as MDH linked oligonucleosides, and methylenecarbonylamino linked
oligonucleosides, also identified as amide-3 linked
oligonucleosides, and methyleneaminocarbonyl linked
oligonucleosides, also identified as amide-4 linked
oligonucleosides, as well as mixed backbone oligomeric compounds
having, for instance, alternating MMI and P.dbd.O or P.dbd.S
linkages are prepared as described in U.S. Pat. Nos. 5,378,825,
5,386,023, 5,489,677, 5,602,240 and 5,610,289.
[0069] Formacetal and thioformacetal linked oligonucleosides are
prepared as described in U.S. Pat. Nos. 5,264,562 and
5,264,564.
[0070] Ethylene oxide linked oligonucleosides are prepared as
described in U.S. Pat. No. 5,223,618.
[0071] In general, RNA synthesis chemistry is based on the
selective incorporation of various protecting groups at strategic
intermediary reactions. Although one of ordinary skill in the art
will understand the use of protecting groups in organic synthesis,
a useful class of protecting groups includes silyl ethers. In
particular bulky silyl ethers are used to protect the 5'-hydroxyl
in combination with an acid-labile orthoester protecting group on
the 2'-hydroxyl. This set of protecting groups is then used with
standard solid-phase synthesis technology. It is important to
lastly remove the acid labile orthoester protecting group after all
other synthetic steps. Moreover, the early use of the silyl
protecting groups during synthesis ensures facile removal when
desired, without undesired deprotection of 2' hydroxyl.
[0072] Following this procedure for the sequential protection of
the 5'-hydroxyl in combination with protection of the 2'-hydroxyl
by protecting groups that are differentially removed and are
differentially chemically labile, RNA oligonucleotides were
synthesized.
[0073] RNA oligonucleotides are synthesized in a stepwise fashion.
Each nucleotide is added sequentially (3'- to 5'-direction) to a
solid support-bound oligonucleotide. The first nucleoside at the
3'-end of the chain is covalently attached to a solid support. The
nucleotide precursor, a ribonucleoside phosphoramidite, and
activator are added, coupling the second base onto the 5'-end of
the first nucleoside. The support is washed and any unreacted
5'-hydroxyl groups are capped with acetic anhydride to yield
5'-acetyl moieties. The linkage is then oxidized to the more stable
and ultimately desired P(V) linkage. At the end of the nucleotide
addition cycle, the 5'-silyl group is cleaved with fluoride. The
cycle is repeated for each subsequent nucleotide.
[0074] Following synthesis, the methyl protecting groups on the
phosphates are cleaved in 30 minutes utilizing 1 M
disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate
(S.sub.2Na.sub.2) in DMF. The deprotection solution is washed from
the solid support-bound oligonucleotide using water. The support is
then treated with 40% methylamine in water for 10 minutes at
55.degree. C. This releases the RNA oligonucleotides into solution,
deprotects the exocyclic amines, and modifies the 2'-groups. The
oligonucleotides can be analyzed by anion exchange HPLC at this
stage.
[0075] The 2'-orthoester groups are the last protecting groups to
be removed. The ethylene glycol monoacetate orthoester protecting
group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is
one example of a useful orthoester protecting group which, has the
following important properties. It is stable to the conditions of
nucleoside phosphoramidite synthesis and oligonucleotide synthesis.
However, after oligonucleotide synthesis the oligonucleotide is
treated with methylamine which not only cleaves the oligonucleotide
from the solid support but also removes the acetyl groups from the
orthoesters. The resulting 2-ethyl-hydroxyl substituents on the
orthoester are less electron withdrawing than the acetylated
precursor. As a result, the modified orthoester becomes more labile
to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is
approximately 10 times faster after the acetyl groups are removed.
Therefore, this orthoester possesses sufficient stability in order
to be compatible with oligonucleotide synthesis and yet, when
subsequently modified, permits deprotection to be carried out under
relatively mild aqueous conditions compatible with the final RNA
oligonucleotide product.
[0076] Additionally, methods of RNA synthesis are well known in the
art (Scaringe, Ph.D. Thesis, University of Colorado, 1996; Scaringe
et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci et
al., J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage et al.,
Tetrahedron Lett., 1981, 22, 1859-1862; Dahl et al., Acta Chem.
Scand., 1990, 44, 639-641; Reddy et al., Tetrahedrom Lett., 1994,
25, 4311-4314; Wincott et al., Nucleic Acids Res., 1995, 23,
2677-2684; Griffin et al., Tetrahedron, 1967, 23, 2301-2313;
Griffin et al., Tetrahedron, 1967, 23, 2315-2331).
[0077] The present invention is also useful for the preparation of
oligomeric compounds incorporating at least one 2'-O-protected
nucleoside. After incorporation and appropriate deprotection the
2'-O-protected nucleoside will be converted to a ribonucleoside at
the position of incorporation. The number and position of the
2-ribonucleoside units in the final oligomeric compound can vary
from one at any site or the strategy can be used to prepare up to a
full 2'-OH modified oligomeric compound. All 2'-O-protecting groups
amenable to the synthesis of oligomeric compounds are included in
the present invention.
[0078] In general a protected nucleoside is attached to a solid
support by for example a succinate linker. Then the oligonucleotide
is elongated by repeated cycles of deprotecting the 5'-terminal
hydroxyl group, coupling of a further nucleoside unit, capping and
oxidation (alternatively sulfurization). In a more frequently used
method of synthesis the completed oligonucleotide is cleaved from
the solid support with the removal of phosphate protecting groups
and exocyclic amino protecting groups by treatment with an ammonia
solution. Then a further deprotection step is normally required for
the more specialized protecting groups used for the protection of
2'-hydroxyl groups which will give the fully deprotected
oligonucleotide.
[0079] A large number of 2'-O-protecting groups have been used for
the synthesis of oligoribonucleotides but over the years more
effective groups have been discovered. The key to an effective
2'-O-protecting group is that it is capable of selectively being
introduced at the 2'-O-position and that it can be removed easily
after synthesis without the formation of unwanted side products.
The protecting group also needs to be inert to the normal
deprotecting, coupling, and capping steps required for
oligoribonucleotide synthesis. Some of the protecting groups used
initially for oligoribonucleotide synthesis included
tetrahydropyran-1-yl and 4-methoxytetrahydropyran-4-yl. These two
groups are not compatible with all 5'-O-protecting groups so
modified versions were used with 5'-DMT groups such as
1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp). Reese has
identified a number of piperidine derivatives (like Fpmp) that are
useful in the synthesis of oligoribonucleotides including
1-((chloro-4-methyl)phenyl)-4'-methoxypiperidin-4-yl (Reese et al.,
Tetrahedron Lett., 1986, 27, 2291). Another approach was to replace
the standard 5'-DMT (dimethoxytrityl) group with protecting groups
that were removed under non-acidic conditions such as levulinyl and
9-fluorenylmethoxycarbonyl. Such groups enable the use of acid
labile 2'-protecting groups for oligoribonucleotide synthesis.
Another more widely used protecting group initially used for the
synthesis of oligoribonucleotides was the t-butyldimethylsilyl
group (Ogilvie et al., Tetrahedron Lett., 1974, 2861; Hakimelahi et
al., Tetrahedron Lett., 1981, 22, 2543; and Jones et al., J. Chem.
Soc. Perkin I., 2762). The 2'-O-protecting groups can require
special reagents for their removal such as for example the
t-butyldimethylsilyl group is normally removed after all other
cleaving/deprotecting steps by treatment of the oligomeric compound
with tetrabutylammonium fluoride (TBAF).
[0080] One group of researchers examined a number of
2'-O-protecting groups (Pitsch, Chimia, 2001, 55, 320-324.) The
group examined fluoride labile and photolabile protecting groups
that are removed using moderate conditions. One photolabile group
that was examined was the (2-(nitrobenzyl)oxy)methyl (nbm)
protecting group (Schwartz et al., Bioorg. Med. Chem. Lett., 1992,
2, 1019.) Other groups examined included a number structurally
related formaldehyde acetal-derived, 2'-O-protecting groups. Also
prepared were a number of related protecting groups for preparing
2'-O-alkylated nucleoside phosphoramidites including
2'-O-((triisopropylsilyl)oxy)methyl
(2'-O--CH.sub.2--O--Si(iPr).sub.3, TOM). One 2'-O-protecting group
that was prepared to be used orthogonally to the TOM group was
2'-O-((R)-1-(2-nitrophenyl)ethyloxy)methyl) ((R)-mnbm).
[0081] Another strategy using a fluoride labile 5'-O-protecting
group (non-acid labile) and an acid labile 2'-O-protecting group
has been reported (Scaringe, Methods, 2001, 23, 206-217). A number
of possible silyl ethers were examined for 5'-O-protection and a
number of acetals and orthoesters were examined for
2'-O-protection. The protection scheme that gave the best results
was 5'-O-silyl ether-2'-ACE
(5'-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether
(DOD)-2'-O-bis(2-acetoxyethoxy)methyl (ACE). This approach uses a
modified phosphoramidite synthesis approach in that some different
reagents are required that are not routinely used for RNA/DNA
synthesis.
[0082] Although a lot of research has focused on the synthesis of
oligoribonucleotides the main RNA synthesis strategies that are
presently being used commercially include
5'-O-DMT-2'-O-t-butyldimethylsilyl (TBDMS),
5'-O-DMT-2'-O-(1(2-fluorophenyl)-4-methoxypiperidin-4-yl) (FPMP),
2'-O-((triisopropylsilyl)oxy)methyl
(2'-O--CH.sub.2--O--Si(iPr).sub.3 (TOM), and the 5'-O-silyl
ether-2'-ACE (5'-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether
(DOD)-2'-O-bis(2-acetoxyethoxy)methyl (ACE). A current list of some
of the major companies currently offering RNA products include
Pierce Nucleic Acid Technologies, Dharmacon Research Inc., Ameri
Biotechnologies Inc., and Integrated DNA Technologies, Inc. One
company, Princeton Separations, is marketing an RNA synthesis
activator advertised to reduce coupling times especially with TOM
and TBDMS chemistries. Such an activator would also be amenable to
the present invention.
[0083] The structures corresponding to these protecting groups are
shown below. ##STR1## ##STR2##
[0084] All of the aforementioned RNA synthesis strategies are
amenable to the present invention. Strategies that would be a
hybrid of the above e.g. using a 5'-protecting group from one
strategy with a 2'-O-protecting from another strategy is also
amenable to the present invention.
[0085] The preparation of ribonucleotides and oligomeric compounds
having at least one ribonucleoside incorporated and all the
possible configurations falling in between these two extremes are
encompassed by the present invention. The corresponding oligomeric
compounds can be hybridized to further oligomeric compounds
including oligoribonucleotides having regions of complementarity to
form double-stranded (duplexed) oligomeric compounds.
[0086] The methods of preparing oligomeric compounds of the present
invention can also be applied in the areas of drug discovery and
target validation.
[0087] After cleavage from the controlled pore glass solid support
and deblocking in concentrated ammonium hydroxide at 55.degree. C.
for 12-16 hours, the oligonucleotides or oligonucleosides are
recovered by precipitation out of 1 M NH.sub.4OAc with >3
volumes of ethanol. Synthesized oligonucleotides were analyzed by
electrospray mass spectroscopy (molecular weight determination) and
by capillary gel electrophoresis and judged to be at least 70% full
length material. The relative amounts of phosphorothioate and
phosphodiester linkages obtained in the synthesis was determined by
the ratio of correct molecular weight relative to the -16 amu
product (+/-32+/-48). For some studies oligonucleotides were
purified by HPLC, as described by Chiang et al., J. Biol. Chem.
1991, 266, 18162-18171. Results obtained with HPLC-purified
material were similar to those obtained with non-HPLC purified
material.
[0088] Oligonucleotides were synthesized via solid phase P(III)
phosphoramidite chemistry on an automated synthesizer capable of
assembling 96 sequences simultaneously in a 96-well format.
Phosphodiester internucleotide linkages were afforded by oxidation
with aqueous iodine. Phosphorothioate internucleotide linkages were
generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one
1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard
base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were
purchased from commercial vendors (e.g. PE-Applied Biosystems,
Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard
nucleosides are synthesized as per standard or patented methods.
They are utilized as base protected beta-cyanoethyldiisopropyl
phosphoramidites.
[0089] Oligonucleotides were cleaved from support and deprotected
with concentrated NH.sub.4OH at elevated temperature (55-60.degree.
C.) for 12-16 hours and the released product then dried in vacuo.
The dried product was then re-suspended in sterile water to afford
a master plate from which all analytical and test plate samples are
then diluted utilizing robotic pipettors.
[0090] The concentration of oligonucleotide in each well was
assessed by dilution of samples and UV absorption spectroscopy. The
full-length integrity of the individual products was evaluated by
capillary electrophoresis (CE) in either the 96-well format
(Beckman P/ACE.TM. MDQ) or, for individually prepared samples, on a
commercial CE apparatus (e.g., Beckman P/ACE.TM. 5000, ABI 270).
Base and backbone composition was confirmed by mass analysis of the
oligomeric compounds utilizing electrospray-mass spectroscopy. All
assay test plates were diluted from the master plate using single
and multi-channel robotic pipettors. Plates were judged to be
acceptable if at least 85% of the oligomeric compounds on the plate
were at least 85% full length.
[0091] For double-stranded compounds of the invention, once
synthesized, the complementary strands are annealed. The single
strands are aliquoted and diluted to a concentration of 50 .mu.M.
Once diluted, 30 .mu.L of each strand is combined with 15 .mu.L of
a 5.times. solution of annealing buffer. The final concentration of
the buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and
2 mM magnesium acetate. The final volume is 75 .mu.L. This solution
is incubated for 1 minute at 90.degree. C. and then centrifuged for
15 seconds. The tube is allowed to sit for 1 hour at 37.degree. C.
at which time the double-stranded compounds are used in
experimentation. The final concentration of the duplexed compound
is 20 .mu.M. This solution can be stored frozen (-20.degree. C.)
and freeze-thawed up to 5 times.
[0092] Once prepared, the double-stranded compounds are evaluated
for their ability to modulate target levels, expression or
function. When cells reach 80% confluency, they are treated with
synthetic double-stranded compounds comprising at least one
oligomeric compound of the invention. For cells grown in 96-well
plates, wells are washed once with 200 .mu.L OPTI-MEM.TM. 1
reduced-serum medium (Gibco BRL) and then treated with 130 .mu.L of
OPTI-MEM.TM.-1 containing 12 .mu.g/mL LIPOFECTIN.TM. (Invitrogen
Corporation, Carlsbad, Calif.) and the desired double stranded
compound at a final concentration of 200 nM. After 5 hours of
treatment, the medium is replaced with fresh medium. Cells are
harvested 16 hours after treatment, at which time RNA is isolated
and target reduction measured by real-time RT-PCR.
[0093] Specific examples of oligomeric compounds useful in this
invention include oligonucleotides containing modified e.g.
non-naturally occurring internucleoside linkages. As defined in
this specification, oligonucleotides having modified
internucleoside linkages include internucleoside linkages that
retain a phosphorus atom and internucleoside linkages that do not
have a phosphorus atom. For the purposes of this specification, and
as sometimes referenced in the art, modified oligonucleotides that
do not have a phosphorus atom in their internucleoside backbone can
also be considered to be oligonucleosides.
[0094] Modified oligonucleotide backbones (internucleoside
linkages) containing a phosphorus atom therein include, for
example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates, 5'-alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters,
selenophosphates and boranophosphates having normal 3'-5' linkages,
2'-5' linked analogs of these, and those having inverted polarity
wherein one or more internucleotide linkages is a 3' to 3', 5' to
5' or 2' to 2' linkage. Oligonucleotides having inverted polarity
comprise a single 3' to 3' linkage at the 3'-most internucleotide
linkage i.e. a single inverted nucleoside residue which may be
abasic (the nucleobase is missing or has a hydroxyl group in place
thereof). Various salts, mixed salts and free acid forms are also
included.
[0095] Representative U.S. patents that teach the preparation of
the above phosphorus-containing linkages include, but are not
limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;
5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;
5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899;
5,721,218; 5,672,697 and 5,625,050.
[0096] In other embodiments of the invention, oligomeric compounds
have one or more phosphorothioate and/or heteroatom internucleoside
linkages, in particular --CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- (known as a methylene
(methylimino) or MMI backbone),
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- (wherein the native
phosphodiester internucleotide linkage is represented as
--O--P(.dbd.O)(OH)--O--CH.sub.2--). The MMI type internucleoside
linkages are disclosed in the above referenced U.S. Pat. No.
5,489,677. Amide internucleoside linkages are disclosed in the
above referenced U.S. Pat. No. 5,602,240.
[0097] Modified oligonucleotide backbones (internucleoside
linkages) that do not include a phosphorus atom therein have
backbones that are formed by short chain alkyl or cycloalkyl
internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl
internucleoside linkages, or one or more short chain heteroatomic
or heterocyclic internucleoside linkages. These include those
having morpholino linkages (formed in part from the sugar portion
of a nucleoside); siloxane backbones; sulfide, sulfoxide and
sulfone backbones; formacetyl and thioformacetyl backbones;
methylene formacetyl and thioformacetyl backbones; riboacetyl
backbones; alkene containing backbones; sulfamate backbones;
methyleneimino and methylenehydrazino backbones; sulfonate and
sulfonamide backbones; amide backbones; and others having mixed N,
O, S and CH.sub.2 component parts.
[0098] Representative U.S. patents that teach the preparation of
the above oligonucleosides include, but are not limited to, U.S.
Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141;
5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677;
5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240;
5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070;
5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and
5,677,439.
[0099] Another group of oligomeric compounds amenable to the
present invention includes oligonucleotide mimetics. The term
mimetic as it is applied to oligonucleotides is intended to include
oligomeric compounds wherein only the furanose ring or both the
furanose ring and the internucleotide linkage are replaced with
novel groups, replacement of only the furanose ring is also
referred to in the art as being a sugar surrogate. The heterocyclic
base moiety or a modified heterocyclic base moiety is maintained
for hybridization with an appropriate target nucleic acid. One such
oligomeric compound, an oligonucleotide mimetic that has been shown
to have excellent hybridization properties, is referred to as a
peptide nucleic acid (PNA). In PNA oligomeric compounds, the
sugar-backbone of an oligonucleotide is replaced with an amide
containing backbone, in particular an aminoethylglycine backbone.
The nucleobases are retained and are bound directly or indirectly
to aza nitrogen atoms of the amide portion of the backbone.
Representative U.S. patents that teach the preparation of PNA
oligomeric compounds include, but are not limited to, U.S. Pat.
Nos. 5,539,082; 5,714,331; and 5,719,262. Teaching of PNA
oligomeric compounds can be found in Nielsen et al., Science, 1991,
254, 1497-1500.
[0100] PNA has been modified to incorporate numerous modifications
since the basic PNA structure was first prepared. The basic
structure is shown below: ##STR3## wherein:
[0101] Bx is a heterocyclic base moiety;
[0102] T.sub.4 is hydrogen, an amino protecting group,
--C(O)R.sub.5, substituted or unsubstituted C.sub.1-C.sub.10 alkyl,
substituted or unsubstituted C.sub.2-C.sub.10 alkenyl, substituted
or unsubstituted C.sub.2-C.sub.10 alkynyl, alkylsulfonyl,
arylsulfonyl, a chemical functional group, a reporter group, a
conjugate group, a D or L .alpha.-amino acid linked via the
.alpha.-carboxyl group or optionally through the .omega.-carboxyl
group when the amino acid is aspartic acid or glutamic acid or a
peptide derived from D, L or mixed D and L amino acids linked
through a carboxyl group, wherein the substituent groups are
selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl,
nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and
alkynyl;
[0103] T.sub.5 is --OH, --N(Z.sub.1)Z.sub.2, R.sub.5, D or L
.alpha.-amino acid linked via the .alpha.-amino group or optionally
through the .omega.-amino group when the amino acid is lysine or
ornithine or a peptide derived from D, L or mixed D and L amino
acids linked through an amino group, a chemical functional group, a
reporter group or a conjugate group;
[0104] Z.sub.1 is hydrogen, C.sub.1-C.sub.6 alkyl, or an amino
protecting group;
[0105] Z.sub.2 is hydrogen, C.sub.1-C.sub.6 alkyl, an amino
protecting group, --C(.dbd.O)--(CH.sub.2).sub.n-J-Z.sub.3, a D or L
.alpha.-amino acid linked via the .alpha.-carboxyl group or
optionally through the .omega.-carboxyl group when the amino acid
is aspartic acid or glutamic acid or a peptide derived from D, L or
mixed D and L amino acids linked through a carboxyl group;
[0106] Z.sub.3 is hydrogen, an amino protecting group,
--C.sub.1-C.sub.6 alkyl, --C(.dbd.O)--CH.sub.3, benzyl, benzoyl, or
--(CH.sub.2).sub.n--N(H)Z.sub.1;
[0107] each J is O, S or NH;
[0108] R.sub.5 is a carbonyl protecting group; and
[0109] n is from 2 to about 450.
[0110] Another class of oligonucleotide mimetic that has been
studied is based on linked morpholino units (morpholino nucleic
acid) having heterocyclic bases attached to the morpholino ring. A
number of linking groups have been reported that link the
morpholino monomeric units in a morpholino nucleic acid. A suitable
class of linking groups have been selected to give a non-ionic
oligomeric compound. The non-ionic morpholino-based oligomeric
compounds are less likely to have undesired interactions with
cellular proteins. Morpholino-based oligomeric compounds are
non-ionic mimics of oligonucleotides which are less likely to form
undesired interactions with cellular proteins (Braasch and Corey,
Biochemistry, 2002, 41, 4503-4510). Morpholino-based oligomeric
compounds are disclosed in U.S. Pat. No. 5,034,506. The morpholino
class of oligomeric compounds have been prepared having a variety
of different linking groups joining the monomeric subunits.
[0111] Morpholino nucleic acids have been prepared having a variety
of different linking groups (L.sub.2) joining the monomeric
subunits. The basic formula is shown below: ##STR4##
[0112] Another class of oligonucleotide mimetic is referred to as
cyclohexenyl nucleic acids (CeNA). The furanose ring normally
present in an DNA/RNA molecule is replaced with a cyclohenyl ring.
CeNA DMT protected phosphoramidite monomers have been prepared and
used for oligomeric compound synthesis following classical
phosphoramidite chemistry. Fully modified CeNA oligomeric compounds
and oligonucleotides having specific positions modified with CeNA
have been prepared and studied (see Wang et al., J. Am. Chem. Soc.,
2000, 122, 8595-8602). In general the incorporation of CeNA
monomers into a DNA chain increases its stability of a DNA/RNA
hybrid. CeNA oligoadenylates formed complexes with RNA and DNA
complements with similar stability to the native complexes. The
study of incorporating CeNA structures into natural nucleic acid
structures was shown by NMR and circular dichroism to proceed with
easy conformational adaptation. Furthermore the incorporation of
CeNA into a sequence targeting RNA was stable to serum and able to
activate E. Coli RNase resulting in cleavage of the target RNA
strand.
[0113] The general formula of CeNA is shown below: ##STR5##
[0114] Another class of oligonucleotide mimetic (anhydrohexitol
nucleic acid) can be prepared from one or more anhydrohexitol
nucleosides (Wouters et al., Bioorg. Med. Chem. Lett., 1999, 9,
1563-1566) and would have the general formula: ##STR6##
[0115] Another group of modifications includes nucleosides having
sugar moieties that are bicyclic thereby locking the sugar
conformational geometry. The most studied of these nucleosides is a
bicyclic sugar moiety having a 4'-CH.sub.2--O-2' bridge. As can be
seen in the structure below the 2'-O-- has been linked via a
methylene group to the 4' carbon. This bridge attaches under the
sugar as shown forcing the sugar ring into a locked 3'-endo
conformation geometry. The .alpha.-L nucleoside has also been
reported wherein the linkage is above the ring and the heterocyclic
base is in the a rather than the .beta.-conformation (see U.S.
Patent Application Publication No. 2003/0087230). The xylo analog
has also been prepared (see U.S. Patent Application Publication No.
2003/0082807). The preferred bridge for a locked nucleic acid (LNA)
is 4'-(--CH.sub.2--).sub.n--O-2' wherein n is 1 or 2. The
literature is confusing when the term locked nucleic acid is used
but in general locked nucleic acids refers to n=1, ENA.TM. refers
to n=2 (U.S. Patent Application Publication No. U.S. 2002/0147332,
Singh et al., Chem. Commun., 1998, 4, 455-456, also see U.S. Pat.
Nos. 6,268,490 and 6,670,461 and U.S. Patent Application
Publication No. U.S. 2003/0207841). However the term locked nucleic
acids can also be used in a more general sense to describe any
bicyclic sugar moiety that has a locked conformation.
[0116] ENA.TM. along with LNA (n=1) have been studied more than the
myriad of other analogs. Oligomeric comounds incorporating LNA and
ENA analogs display very high duplex thermal stabilities with
complementary DNA and RNA (Tm=+3 to +10 C), stability towards
3'-exonucleolytic degradation and good solubility properties.
[0117] The basic structure of LNA showing the bicyclic ring system
is shown below: ##STR7##
[0118] The conformations of LNAs determined by 2D NMR spectroscopy
have shown that the locked orientation of the LNA nucleotides, both
in single-stranded LNA and in duplexes, constrains the phosphate
backbone in such a way as to introduce a higher population of the
N-type conformation (Petersen et al., J. Mol. Recognit., 2000, 13,
44-53). These conformations are associated with improved stacking
of the nucleobases (Wengel et al., Nucleosides Nucleotides, 1999,
18, 1365-1370).
[0119] LNA has been shown to form exceedingly stable LNA:LNA
duplexes (Koshkin et al., J. Am. Chem. Soc., 1998, 120,
13252-13253). LNA:LNA hybridization was shown to be the most
thermally stable nucleic acid type duplex system, and the
RNA-mimicking character of LNA was established at the duplex level.
Introduction of 3 LNA monomers (T or A) significantly increased
melting points (Tm=+15/+11) toward DNA complements. The
universality of LNA-mediated hybridization has been stressed by the
formation of exceedingly stable LNA:LNA duplexes. The RNA-mimicking
of LNA was reflected with regard to the N-type conformational
restriction of the monomers and to the secondary structure of the
LNA:RNA duplex.
[0120] LNAs also form duplexes with complementary DNA, RNA or LNA
with high thermal affinities. Circular dichroism (CD) spectra show
that duplexes involving fully modified LNA (esp. LNA:RNA)
structurally resemble an A-form RNA:RNA duplex. Nuclear magnetic
resonance (NMR) examination of an LNA:DNA duplex confirmed the
3'-endo conformation of an LNA monomer. Recognition of
double-stranded DNA has also been demonstrated suggesting strand
invasion by LNA. Studies of mismatched sequences show that LNAs
obey the Watson-Crick base pairing rules with generally improved
selectivity compared to the corresponding unmodified reference
strands.
[0121] Novel types of LNA-oligomeric compounds, as well as the
LNAs, are useful in a wide range of diagnostic and therapeutic
applications. Among these are antisense applications, PCR
applications, strand-displacement oligomers, substrates for nucleic
acid polymerases and generally as nucleotide based drugs.
[0122] Potent and nontoxic antisense oligonucleotides containing
LNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci.
U.S.A., 2000, 97, 5633-5638.) The authors have demonstrated that
LNAs confer several desired properties to antisense agents. LNA/DNA
copolymers were not degraded readily in blood serum and cell
extracts. LNA/DNA copolymers exhibited potent antisense activity in
assay systems as disparate as G-protein-coupled receptor signaling
in living rat brain and detection of reporter genes in Escherichia
coli. LIPOFECTIN.TM.-mediated efficient delivery of LNA into living
human breast cancer cells has also been accomplished.
[0123] The synthesis and preparation of the LNA monomers adenine,
cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along
with their oligomerization, and nucleic acid recognition properties
have been described (Koshkin et al., Tetrahedron, 1998, 54,
3607-3630). LNAs and preparation thereof are also described in WO
98/39352 and WO 99/14226.
[0124] The first analogs of LNA, phosphorothioate-LNA and
2'-thio-LNAs, have also been prepared (Kumar et al., Bioorg. Med.
Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside
analogs containing oligodeoxyribonucleotide duplexes as substrates
for nucleic acid polymerases has also been described (PCT
International Application WO 98-DK393 19980914). Furthermore,
synthesis of 2'-amino-LNA, a novel conformationally restricted
high-affinity oligonucleotide analog with a handle has been
described in the art (Singh et al., J. Org. Chem., 1998, 63,
10035-10039). In addition, 2'-Amino- and 2`-methylamino-LNA`s have
been prepared and the thermal stability of their duplexes with
complementary RNA and DNA strands has been previously reported.
[0125] Some oligonucleotide mimetics have been prepared to incude
bicyclic and tricyclic nucleoside analogs having the formulas
(amidite monomers shown): ##STR8##
[0126] (see Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439;
Steffens et al., J. Am. Chem. Soc., 1999, 121, 3249-3255; and
Renneberg et al., J. Am. Chem. Soc., 2002, 124, 5993-6002). These
modified nucleoside analogs have been oligomerized using the
phosphoramidite approach and the resulting oligomeric compounds
containing tricyclic nucleoside analogs have shown increased
thermal stabilities (Tms) when hybridized to DNA, RNA and itself.
Oligomeric compounds containing bicyclic nucleoside analogs have
shown thermal stabilities approaching that of DNA duplexes.
[0127] Another class of oligonucleotide mimetic is referred to as
phosphonomonoester nucleic acid and incorporates a phosphorus group
in the backbone. This class of olignucleotide mimetic is reported
to have useful physical and biological and pharmacological
properties in the areas of inhibiting gene expression (antisense
oligonucleotides, ribozymes, sense oligonucleotides and
triplex-forming oligonucleotides), as probes for the detection of
nucleic acids and as auxiliaries for use in molecular biology.
[0128] The general formula (for definitions of Markush variables
see: U.S. Pat. Nos. 5,874,553 and 6,127,346) is shown below.
##STR9##
[0129] Another oligonucleotide mimetic has been reported wherein
the furanosyl ring has been replaced by a cyclobutyl moiety.
[0130] Modified sugars: Oligomeric compounds of the invention may
also contain one or more substituted sugar moieties. These
oligomeric compounds comprise a sugar substituent group selected
from: OH; F; O--, S--, or N-alkyl; O--, S--, or N-alkenyl; O--, S--
or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and
alkynyl may be substituted or unsubstituted C.sub.1 to C.sub.10
alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Particularly
suitable are O((CH.sub.2).sub.nO).sub.mCH.sub.3,
O(CH.sub.2).sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON((CH.sub.2).sub.nCH.sub.3).sub.2, where n and m
are from 1 to about 10. Some oligonucleotides comprise a sugar
substituent group selected from: C.sub.1 to C.sub.10 lower alkyl,
substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl,
O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3,
OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2,
N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl,
aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving
group, a reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. One modification
includes 2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub.3, also
known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv.
Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. Another
modification includes 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples hereinbelow, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.3).sub.2.
[0131] Other sugar substituent groups include methoxy
(--O--CH.sub.3), aminopropoxy
(--OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), allyl
(--CH.sub.2--CH.dbd.CH.sub.2), --O-allyl
(--O--CH.sub.2--CH.dbd.CH.sub.2) and fluoro (F). 2'-Sugar
substituent groups may be in the arabino (up) position or ribo
(down) position. One 2'-arabino modification is 2'-F. Similar
modifications may also be made at other positions on the oligomeric
compound, particularly the 3' position of the sugar on the 3'
terminal nucleoside or in 2'-5' linked oligonucleotides and the 5'
position of 5' terminal nucleotide. Oligomeric compounds may also
have sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl sugar. Representative U.S. patents that teach the
preparation of such modified sugar structures include, but are not
limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080;
5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134;
5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053;
5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and
5,700,920.
[0132] Representative sugar substituent groups include groups of
formula I.sub.a or II.sub.a: ##STR10##
[0133] each R.sub.s, R.sub.t, R.sub.u and R.sub.v is,
independently, hydrogen, C(O)R.sub.w, substituted or unsubstituted
C.sub.1-C.sub.10 alkyl, substituted or unsubstituted
C.sub.2-C.sub.10 alkenyl, substituted or unsubstituted
C.sub.2-C.sub.10 alkynyl, alkylsulfonyl, arylsulfonyl, a chemical
functional group or a conjugate group, wherein the substituent
groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl,
phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and
alkynyl;
[0134] or optionally, R.sub.u and R.sub.v, together form a
phthalimido moiety with the nitrogen atom to which they are
attached;
[0135] each R.sub.w is, independently, substituted or unsubstituted
C.sub.1-C.sub.10 alkyl, trifluoromethyl, cyanoethyloxy, methoxy,
ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy,
2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy,
butyryl, iso-butyryl, phenyl or aryl;
[0136] R.sub.k is hydrogen, a nitrogen protecting group or
--R.sub.x--R.sub.y;
[0137] R.sub.p is hydrogen, a nitrogen protecting group or
--R.sub.x--R.sub.y;
[0138] R.sub.x is a bond or a linking moiety;
[0139] R.sub.y is a chemical functional group, a conjugate group or
a solid support medium; [0140] each R.sub.m and R.sub.n is,
independently, H, a nitrogen protecting group, substituted or
unsubstituted C.sub.1-C.sub.10 alkyl, substituted or unsubstituted
C.sub.2-C.sub.10 alkenyl, substituted or unsubstituted
C.sub.2-C.sub.10 alkynyl, wherein the substituent groups are
selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl,
nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, alkynyl;
NH.sub.3.sup.+, N(R.sub.u)(R.sub.v) guanidino and acyl where said
acyl is an acid amide or an ester;
[0141] or R.sub.m and R.sub.n, together, are a nitrogen protecting
group, are joined in a ring structure that optionally includes an
additional heteroatom selected from N and O or are a chemical
functional group;
[0142] R.sub.i is OR.sub.z, SR.sub.z, or N(R.sub.z).sub.2;
[0143] each R.sub.z is, independently, H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 haloalkyl, C(.dbd.NH)N(H)R.sub.u,
C(.dbd.O)N(H)R.sub.u or OC(.dbd.O)N(H)R.sub.u;
[0144] R.sub.f, R.sub.g and R.sub.h comprise a ring system having
from about 4 to about 7 carbon atoms or having from about 3 to
about 6 carbon atoms and 1 or 2 heteroatoms wherein said
heteroatoms are selected from oxygen, nitrogen and sulfur and
wherein said ring system is aliphatic, unsaturated aliphatic,
aromatic, or saturated or unsaturated heterocyclic;
[0145] R.sub.j is alkyl or haloalkyl having 1 to about 10 carbon
atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2
to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms,
N(R.sub.k)(R.sub.m)OR.sub.k, halo, SR.sub.k or CN;
[0146] m.sub.a is 1 to about 10;
[0147] each mb is, independently, 0 or 1;
[0148] mc is 0 or an integer from 1 to 10;
[0149] md is an integer from 1 to 10;
[0150] me is from 0, 1 or 2; and
[0151] provided that when mc is 0, md is greater than 1.
[0152] Representative substituents groups are disclosed in U.S.
patent application Ser. No. 09/130,973.
[0153] Representative cyclic substituent groups are disclosed in
U.S. patent application Ser. No. 09/123,108.
[0154] Particular sugar substituent groups include
O((CH.sub.2).sub.nO).sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON((CH.sub.2).sub.nCH.sub.3)).sub.2, where n and m
are from 1 to about 10.
[0155] Representative guanidino substituent groups are disclosed in
U.S. patent application Ser. No. 09/349,040.
[0156] Representative acetamido substituent groups are disclosed in
U.S. Pat. No. 6,147,200.
[0157] Representative dimethylaminoethyloxyethyl substituent groups
are disclosed in International Patent Application
PCT/US99/17895.
[0158] Chimeric oligonucleotides, oligonucleosides or mixed
oligonucleotides/oligonucleosides of the invention can be of
several different types. These include a first type wherein the
"gap" segment of linked nucleosides is positioned between 5' and 3'
"wing" segments of linked nucleosides and a second "open end" type
wherein the "gap" segment is located at either the 3' or the 5'
terminus of the oligomeric compound. Oligonucleotides of the first
type are also known in the art as "gapmers" or gapped
oligonucleotides. Oligonucleotides of the second type are also
known in the art as "hemimers" or "wingmers."
(2'-O-Me)-(2'-deoxy)-(2'-O-Me) Chimeric Phosphorothioate
Oligonucleotides
[0159] Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate
and 2'-deoxy phosphorothioate oligonucleotide segments are
synthesized using an Applied Biosystems automated DNA synthesizer
Model 394, as above. Oligonucleotides are synthesized using the
automated synthesizer and
2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA
portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for
5' and 3' wings. The standard synthesis cycle is modified by
incorporating coupling steps with increased reaction times for the
5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite. The fully
protected oligonucleotide is cleaved from the support and
deprotected in concentrated ammonia (NH.sub.4OH) for 12-16 hr at
55.degree. C. The deprotected oligo is then recovered by an
appropriate method (precipitation, column chromatography, volume
reduced in vacuo and analyzed spetrophotometrically for yield and
for purity by capillary electrophoresis and by mass
spectrometry.
(2'-O-(2-Methoxyethyl))-(2'-deoxy)-(2'-O-(Methoxyethyl)) Chimeric
Phosphorothioate Oligonucleotides
[0160] (2'-O-(2-methoxyethyl))-(2'-deoxy)-(-2'-O-(methoxyethyl))
chimeric phosphorothioate oligonucleotides were prepared as per the
procedure above for the 2'-O-methyl chimeric oligonucleotide, with
the substitution of 2'-O-(methoxyethyl) amidites for the
2'-O-methyl amidites.
(2'-O-(2-Methoxyethyl)Phosphodiester)-(2'-deoxy
Phosphorothioate)-(2'-O-(2-Methoxyethyl) Phosphodiester) Chimeric
Oligonucleotides
[0161] (2'-O-(2-methoxyethyl phosphodiester)--(2'-deoxy
phosphorothioate)--(2'-O-(methoxyethyl) phosphodiester) chimeric
oligonucleotides are prepared as per the above procedure for the
2'-O-methyl chimeric oligonucleotide with the substitution of
2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites,
oxidation with iodine to generate the phosphodiester
internucleotide linkages within the wing portions of the chimeric
structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one
1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate
internucleotide linkages for the center gap.
[0162] Other chimeric oligonucleotides, chimeric oligonucleosides
and mixed chimeric oligonucleotides/oligonucleosides are
synthesized according to U.S. Pat. No. 5,623,065.
[0163] The terms used to describe the conformational geometry of
homoduplex nucleic acids are "A Form" for RNA and "B Form" for DNA.
The respective conformational geometry for RNA and DNA duplexes was
determined from X-ray diffraction analysis of nucleic acid fibers
(Arnott et al., Biochem. Biophys. Res. Comm., 1970, 47, 1504). In
general, RNA:RNA duplexes are more stable and have higher melting
temperatures (Tms) than DNA:DNA duplexes (Sanger et al., Principles
of Nucleic Acid Structure, 1984, Springer-Verlag; New York, N.Y.;
Lesnik et al., Biochemistry, 1995, 34, 10807-40815; Conte et al.,
Nucleic Acids Res., 1997, 25, 2627-2634). The increased stability
of RNA has been attributed to several structural features, most
notably the improved base stacking interactions that result from an
A-form geometry (Searle et al., Nucleic Acids Res., 1993, 21,
2051-2056). The presence of the 2' hydroxyl in RNA biases the sugar
toward a C3' endo pucker, i.e., also designated as Northern pucker,
which causes the duplex to favor the A-form geometry. In addition,
the 2' hydroxyl groups of RNA can form a network of water mediated
hydrogen bonds that help stabilize the RNA duplex (Egli et al.,
Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy
nucleic acids prefer a C2' endo sugar pucker, i.e., also known as
Southern pucker, which is thought to impart a less stable B-form
geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure,
Springer-Verlag, New York, N.Y.). As used herein, B-form geometry
is inclusive of both C2'-endo pucker and 04'-endo pucker. This is
consistent with Berger, et. al., Nucleic Acids Research, 1998, 26,
2473-2480, who pointed out that in considering the furanose
conformations which give rise to B-form duplexes consideration
should also be given to a 04'-endo pucker contribution.
[0164] DNA:RNA hybrid duplexes, however, are usually less stable
than pure RNA:RNA duplexes, and depending on their sequence may be
either more or less stable than DNA:DNA duplexes (Searle et al.,
Nucleic Acids Res., 1993, 21, 2051-2056). The structure of a hybrid
duplex is intermediate between A- and B-form geometries, which may
result in poor stacking interactions (Lane et al., Eur. J.
Biochem., 1993, 215, 297-306; Fedoroff et al., J. Mol. Biol., 1993,
233, 509-523; Gonzalez et al., Biochemistry, 1995, 34, 4969-4982;
Horton et al., J. Mol. Biol., 1996, 264, 521-533). The stability of
the duplex formed between a target RNA and a synthetic sequence is
central to therapies such as, but not limited to, antisense
mechanisms, including RNase H-mediated and RNA interference
mechanisms, as these mechanisms involved the hybridization of a
synthetic sequence strand to an RNA target strand. In the case of
RNase H, effective inhibition of the mRNA requires that the
antisense sequence achieve at least a threshold of
hybridization.
[0165] One routinely used method of modifying the sugar puckering
is the substitution of the sugar at the 2'-position with a
substituent group that influences the sugar geometry. The influence
on ring conformation is dependent on the nature of the substituent
at the 2'-position. A number of different substituents have been
studied to determine their sugar puckering effect. For example,
2'-halogens have been studied showing that the 2'-fluoro derivative
exhibits the largest population (65%) of the C3'-endo form, and the
2'-iodo exhibits the lowest population (7%). The populations of
adenosine (2'-OH) versus deoxyadenosine (2'-H) are 36% and 19%,
respectively. Furthermore, the effect of the 2'-fluoro group of
adenosine dimers
(2'-deoxy-2'-fluoroadenosine-2'-deoxy-2'-fluoro-adenosine) is also
correlated to the stabilization of the stacked conformation.
[0166] As expected, the relative duplex stability can be enhanced
by replacement of 2'-OH groups with 2'-F groups thereby increasing
the C3'-endo population. It is assumed that the highly polar nature
of the 2'-F bond and the extreme preference for C3'-endo puckering
may stabilize the stacked conformation in an A-form duplex. Data
from UV hypochromicity, circular dichroism, and .sup.1H NMR also
indicate that the degree of stacking decreases as the
electronegativity of the halo substituent decreases. Furthermore,
steric bulk at the 2'-position of the sugar moiety is better
accommodated in an A-form duplex than a B-form duplex. Thus, a
2'-substituent on the 3'-terminus of a dinucleoside monophosphate
is thought to exert a number of effects on the stacking
conformation: steric repulsion, furanose puckering preference,
electrostatic repulsion, hydrophobic attraction, and hydrogen
bonding capabilities. These substituent effects are thought to be
determined by the molecular size, electronegativity, and
hydrophobicity of the substituent. Melting temperatures of
complementary strands is also increased with the 2'-substituted
adenosine diphosphates. It is not clear whether the 3'-endo
preference of the conformation or the presence of the substituent
is responsible for the increased binding. However, greater overlap
of adjacent bases (stacking) can be achieved with the 3'-endo
conformation.
[0167] Nucleoside conformation is influenced by various factors
including substitution at the 2', 3' or 4'-positions of the
pentofuranosyl sugar. Electronegative substituents generally prefer
the axial positions, while sterically demanding substituents
generally prefer the equatorial positions (Principles of Nucleic
Acid Structure, Wolfgang Sanger, 1984, Springer-Verlag.)
Modification of the 2' position to favor the 3'-endo conformation
can be achieved while maintaining the 2'-OH as a recognition
element, as illustrated in FIG. 2, below (Gallo et al.,
Tetrahedron, 2001, 57, 5707-5713. Harry-O'kuru et al., J. Org.
Chem., 1997, 62, 1754-1759 and Tang et al., J. Org. Chem., 1999,
64, 747-754). Alternatively, preference for the 3'-endo
conformation can be achieved by deletion of the 2'-OH as
exemplified by 2'deoxy-2'F-nucleosides (Kawasaki et al., J. Med.
Chem., 1993, 36, 831-841), which adopts the 3'-endo conformation
positioning the electronegative fluorine atom in the axial
position. Other modifications of the ribose ring, for example
substitution at the 4'-position to give 4'-F modified nucleosides
(Guillerm et al., Bioorg. Med. Chem. Lett., 1995, 5, 1455-1460 and
Owen et al., J. Org. Chem., 1976, 41, 3010-3017), or for example
modification to yield methanocarba nucleoside analogs (Jacobson et
al., J. Med. Chem. Lett., 2000, 43, 2196-2203 and Lee et al.,
Bioorg. Med. Chem. Lett., 2001, 11, 1333-1337) also induce
preference for the 3'-endo conformation.
[0168] In one aspect of the present invention oligomeric compounds
include nucleosides synthetically modified to induce a 3'-endo
sugar conformation. A nucleoside can incorporate synthetic
modifications of the heterocyclic base, the sugar moiety or both to
induce a desired 3'-endo sugar conformation. These modified
nucleosides are used to mimic RNA-like nucleosides so that
particular properties of an oligomeric compound can be enhanced
while maintaining the desirable 3'-endo conformational geometry
(see Scheme 1). There is an apparent preference for an RNA type
duplex (A form helix, predominantly 3'-endo) as a requirement (e.g.
trigger) of RNA interference which is supported in part by the fact
that duplexes composed of 2'-deoxy-2'-F-nucleosides appears
efficient in triggering RNAi response in the C. elegans system.
Properties that are enhanced by using more stable 3'-endo
nucleosides include but aren't limited to modulation of
pharmacokinetic properties through modification of protein binding,
protein off-rate, absorption and clearance; modulation of nuclease
stability as well as chemical stability; modulation of the binding
affinity and specificity of the oligomer (affinity and specificity
for enzymes as well as for complementary sequences); and increasing
efficacy of RNA cleavage. The present invention provides oligomeric
compounds designed to act as triggers of RNAi having one or more
nucleosides modified in such a way as to favor a C3'-endo type
conformation. ##STR11##
[0169] Along similar lines, oligomeric triggers of RNAi response
might be composed of one or more nucleosides modified in such a way
that conformation is locked into a C3'-endo type conformation, i.e.
Locked Nucleic Acid (LNA, Singh et al, Chem. Commun., 1998, 4,
455-456), and ethylene bridged Nucleic Acids (ENA, Morita et al,
Bioorg. Med. Chem. Lett., 2002, 12, 73-76). Examples of modified
nucleosides amenable to the present invention are shown below.
These examples are meant to be representative and not exhaustive.
##STR12##
[0170] Oligomeric compounds may also include nucleobase (often
referred to in the art simply as "base" or "heterocyclic base
moiety") modifications or substitutions. As used herein,
"unmodified" or "natural" nucleobases include the purine bases
adenine (A) and guanine (G), and the pyrimidine bases thymine (T),
cytosine (C) and uracil (U). Modified nucleobases also referred
herein as heterocyclic base moieties include other synthetic and
natural nucleobases such as 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and
cytosine, 5-propynyl (--C.ident.C--CH.sub.3) uracil and cytosine
and other alkynyl derivatives of pyrimidine bases, 6-azo uracil,
cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,
8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other
8-substituted adenines and guanines, 5-halo particularly 5-bromo,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,
8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine
and 3-deazaguanine and 3-deazaadenine.
[0171] Heterocyclic base moieties may also include those in which
the purine or pyrimidine base is replaced with other heterocycles,
for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and
2-pyridone. Some nucleobases include those disclosed in U.S. Pat.
No. 3,687,808, those disclosed in The Concise Encyclopedia Of
Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I.,
ed. John Wiley & Sons, 1990, those disclosed by Englisch et
al., Angewandte Chemie, International Edition, 1991, 30, 613, and
those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research
and Applications, pages 289-302, Crooke and Lebleu, Eds., CRC
Press, 1993. Certain of these nucleobases are particularly useful
for increasing the binding affinity of the oligomeric compounds of
the invention. These include 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and O-6 substituted purines,
including 2 aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. 5-methylcytosine substitutions have been shown
to increase nucleic acid duplex stability by 0.6-1.2.degree. C.
(Sanghvi et al., Eds., Antisense Research and Applications, CRC
Press, Boca Raton, 1993, pp. 276-278) and are presently preferred
base substitutions, even more particularly when combined with
2'-O-methoxyethyl sugar modifications.
[0172] In one aspect of the present invention oligomeric compounds
are prepared having polycyclic heterocyclic compounds in place of
one or more heterocyclic base moieties. A number of tricyclic
heterocyclic compounds have been previously reported. These
compounds are routinely used in antisense applications to increase
the binding properties of the modified strand to a target strand.
The most studied modifications are targeted to guanosines hence
they have been termed G-clamps or cytidine analogs. Many of these
polycyclic heterocyclic compounds have the general formula:
##STR13##
[0173] Representative cytosine analogs that make 3 hydrogen bonds
with a guanosine in a second strand include
1,3-diazaphenoxazine-2-one (R.sub.10=O, R.sub.11-R.sub.14=H)
(Kurchavov et al., Nucleosides and Nucleotides, 1997, 16,
1837-1846), 1,3-diazaphenothiazine-2-one (R.sub.10=S,
R.sub.11-R.sub.14.dbd.H), (Lin et al., J. Am. Chem. Soc., 1995,
117, 3873-3874) and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one
(R.sub.10=O, R.sub.11-R.sub.14=F) (Wang et al., Tetrahedron Lett.,
1998, 39, 8385-8388). When incorporated into oligonucleotides,
these base modifications were shown to hybridize with complementary
guanine and the latter was also shown to hybridize with adenine and
to enhance helical thermal stability by extended stacking
interactions (also see U.S. Patent Application Publications
20030207804 and 20030175906).
[0174] Helix-stabilizing properties have been observed when a
cytosine analog/substitute has an aminoethoxy moiety attached to
the rigid 1,3-diazaphenoxazine-2-one scaffold (R.sub.10=O,
R.sub.11=--O--(CH.sub.2).sub.2--NH.sub.2, R.sub.12-14.dbd.H) (Lin
et al., J. Am. Chem. Soc., 1998, 120, 8531-8532). Binding studies
demonstrated that a single incorporation could enhance the binding
affinity of a model oligonucleotide to its complementary target DNA
or RNA with a .DELTA.T.sub.m of up to 18.degree. relative to
5-methyl cytosine (dC5.sup.me), which is the highest known affinity
enhancement for a single modification. On the other hand, the gain
in helical stability does not compromise the specificity of the
oligonucleotides. The T.sub.m data indicate an even greater
discrimination between the perfect match and mismatched sequences
compared to dC5.sup.me. It was suggested that the tethered amino
group serves as an additional hydrogen bond donor to interact with
the Hoogsteen face, namely the O6, of a complementary guanine
thereby forming 4 hydrogen bonds. This means that the increased
affinity of G-clamp is mediated by the combination of extended base
stacking and additional specific hydrogen bonding.
[0175] Tricyclic heterocyclic compounds and methods of using them
that are amenable to the present invention are disclosed in U.S.
Pat. No. 6,028,183, and U.S. Pat. No. 6,007,992.
[0176] The enhanced binding affinity of the phenoxazine derivatives
together with their sequence specificity makes them valuable
nucleobase analogs for the development of more potent
antisense-based drugs. In fact, promising data have been derived
from in vitro experiments demonstrating that heptanucleotides
containing phenoxazine substitutions can activate RNaseH, enhance
cellular uptake and exhibit an increased antisense activity (Lin et
al., J. Am. Chem. Soc., 1998, 120, 8531-8532). The activity
enhancement was even more pronounced in case of G-clamp, as a
single substitution was shown to significantly improve the in vitro
potency of a 20mer 2'-deoxyphosphorothioate oligonucleotides
(Flanagan et al., Proc. Natl. Acad. Sci. USA, 1999, 96,
3513-3518).
[0177] Modified polycyclic heterocyclic compounds useful as
heterocyclic bases are disclosed in but not limited to, the above
noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205;
5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257;
5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;
5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269;
5,750,692; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, and U.S.
Patent Application Publication 20030158403.
[0178] One substitution that can be appended to the oligomeric
compounds of the invention involves the linkage of one or more
moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the resulting oligomeric
compounds. In one embodiment such modified oligomeric compounds are
prepared by covalently attaching conjugate groups to functional
groups such as hydroxyl or amino groups. Conjugate groups of the
invention include intercalators, reporter molecules, polyamines,
polyamides, polyethylene glycols, polyethers, groups that enhance
the pharmacodynamic properties of oligomers, and groups that
enhance the pharmacokinetic properties of oligomers. Typical
conjugates groups include cholesterols, carbohydrates, lipids,
phospholipids, biotin, phenazine, folate, phenanthridine,
anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and
dyes. Groups that enhance the pharmacodynamic properties, in the
context of this invention, include groups that improve oligomer
uptake, enhance oligomer resistance to degradation, and/or
strengthen hybridization with RNA. Groups that enhance the
pharmacokinetic properties, in the context of this invention,
include groups that improve oligomer uptake, distribution,
metabolism or excretion. Representative conjugate groups are
disclosed in International Patent Application PCT/US92/09196.
Conjugate moieties include but are not limited to lipid moieties
such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad.
Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al.,
Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,
hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,
660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3,
2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,
1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or
undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10,
1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;
Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,
e.g., di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923-937).
[0179] The oligomeric compounds of the invention may also be
conjugated to active drug substances, for example, aspirin,
warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen,
ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine,
2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a
benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a
barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an
antibacterial or an antibiotic. Oligonucleotide-drug conjugates and
their preparation are described in U.S. patent application Ser. No.
09/334,130.
[0180] Representative U.S. patents that teach the preparation of
such oligonucleotide conjugates include, but are not limited to,
U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465;
5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731;
5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603;
5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;
4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582;
4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963;
5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250;
5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463;
5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142;
5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928
and 5,688,941.
[0181] Oligomeric compounds used in the compositions of the present
invention can also be modified to have one or more stabilizing
groups that are generally attached to one or both termini of
oligomeric compounds to enhance properties such as for example
nuclease stability. Included in stabilizing groups are cap
structures. By "cap structure or terminal cap moiety" is meant
chemical modifications, which have been incorporated at either
terminus of oligonucleotides (see for example, WO 97/26270). These
terminal modifications protect the oligomeric compounds having
terminal nucleic acid molecules from exonuclease degradation, and
can help in delivery and/or localization within a cell. The cap can
be present at the 5'-terminus (5'-cap) or at the 3'-terminus
(3'-cap) or can be present on both termini. For double-stranded
oligomeric compounds, the cap may be present at either or both
termini of either strand. In non-limiting examples, the 5'-cap
includes inverted abasic residue (moiety), 4',5'-methylene
nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio
nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide;
L-nucleotides; alpha-nucleotides; modified base nucleotide;
phosphorodithioate linkage; threo-pentofuranosyl nucleotide;
acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl
nucleotide; acyclic 3,5-dihydroxypentyl riucleotide, 3'-3'-inverted
nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted
nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol
phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl
phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate;
or bridging or non-bridging methylphosphonate moiety (International
PCT publication No. WO 97/26270).
[0182] Particularly preferred 3'-cap structures of the present
invention include, for example 4',5'-methylene nucleotide;
1-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide,
carbocyclic nucleotide; 5'-amino-alkyl phosphate;
1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate;
6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl
phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide;
alpha-nucleotide; modified base nucleotide; phosphorodithioate;
threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide;
3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide,
5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety;
5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate;
5'-amino; bridging and/or non-bridging 5'-phosphoramidate,
phosphorothioate and/or phosphorodithioate, bridging or non
bridging methylphosphonate and 5'-mercapto moieties (for more
details see Beaucage and Tyer, 1993, Tetrahedron 49, 1925).
[0183] Further 3' and 5'-stabilizing groups that can be used to cap
one or both ends of an oligomeric compound to impart nuclease
stability include those disclosed in WO 03/004602.
[0184] It is not necessary for all positions in an oligomeric
compound to be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
oligomeric compound or even at a single monomeric subunit such as a
nucleoside within a oligomeric compound. The present invention also
includes oligomeric compounds which are chimeric oligomeric
compounds. "Chimeric" oligomeric compounds or "chimeras," in the
context of this invention, are oligomeric compounds that contain
two or more chemically distinct regions, each made up of at least
one monomer unit, i.e., a nucleotide in the case of a nucleic acid
based oligomer.
[0185] Chimeric oligomeric compounds typically contain at least one
region modified so as to confer increased resistance to nuclease
degradation, increased cellular uptake, and/or increased binding
affinity for the target nucleic acid. An additional region of the
oligomeric compound may serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids. By way of example, an
oligomeric compound may be designed to comprise a region that
serves as a substrate for RNase H. RNase H is a cellular
endonuclease which cleaves the RNA strand of an RNA:DNA duplex.
Activation of RNase H by an oligomeric compound having a cleavage
region, therefore, results in cleavage of the RNA target, thereby
enhancing the efficiency of the oligomeric compound. Consequently,
comparable results can often be obtained with shorter oligomeric
compounds having substrate regions when chimeras are used, compared
to for example phosphorothioate deoxyoligonucleotides hybridizing
to the same target region. Cleavage of the RNA target can be
routinely detected by gel electrophoresis and, if necessary,
associated nucleic acid hybridization techniques known in the
art.
[0186] Chimeric oligomeric compounds of the invention may be formed
as composite structures of two or more oligonucleotides,
oligonucleotide mimics, oligonucleotide analogs, oligonucleosides
and/or oligonucleotide mimetics as described above. Such oligomeric
compounds have also been referred to in the art as hybrids,
hemimers, gapmers or inverted gapmers. Representative U.S. patents
that teach the preparation of such hybrid structures include, but
are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007;
5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;
5,652,355; 5,652,356; and 5,700,922.
[0187] The conformation of modified nucleosides and their oligomers
can be estimated by various methods such as molecular dynamics
calculations, nuclear magnetic resonance spectroscopy and CD
measurements. Hence, modifications predicted to induce RNA-like
conformations (A-form duplex geometry in an oligomeric context),
are useful in the oligomeric compounds of the present invention.
The synthesis of modified nucleosides amenable to the present
invention are known in the art (see for example, Chemistry of
Nucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend, 1988,
Plenum Press.)
[0188] In one aspect, the present invention is directed to
oligomeric compounds that are designed to have enhanced properties
compared to native RNA. One method to design optimized or enhanced
oligomeric compounds involves each nucleoside of the selected
sequence being scrutinized for possible enhancing modifications.
One modification would be the replacement of one or more RNA
nucleosides with nucleosides that have the same 3'-endo
conformational geometry. Such modifications can enhance chemical
and nuclease stability relative to native RNA while at the same
time being much cheaper and easier to synthesize and/or incorporate
into an oligonucleotide. The sequence can be further divided into
regions and the nucleosides of each region evaluated for enhancing
modifications that can be the result of a chimeric configuration.
Consideration is also given to the 5' and 3'-termini as there are
often advantageous modifications that can be made to one or more of
the terminal nucleosides. The oligomeric compounds of the present
invention may include at least one 5'-modified phosphate group on a
single strand or on at least one 5'-position of a double-stranded
sequence or sequences. Other modifications considered are
internucleoside linkages, conjugate groups, substitute sugars or
bases, substitution of one or more nucleosides with nucleoside
mimetics and any other modification that can enhance the desired
property of the oligomeric compound.
[0189] One synthetic 2'-modification that imparts increased
nuclease resistance and a very high binding affinity to nucleotides
is the 2-methoxyethoxy (2'-MOE, 2'-OCH.sub.2CH.sub.2OCH.sub.3) side
chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). One
of the immediate advantages of the 2'-MOE substitution is the
improvement in binding affinity, which is greater than many similar
2' modifications such as O-methyl, O-propyl, and O-aminopropyl.
Oligonucleotides having the 2'-O-methoxyethyl substituent also have
been shown to be antisense inhibitors of gene expression with
promising features for in vivo use (Martin, Helv. Chim. Acta, 1995,
78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et
al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al.,
Nucleosides Nucleotides, 1997, 16, 917-926). Relative to DNA, the
oligonucleotides having the 2'-MOE modification displayed improved
RNA affinity and higher nuclease resistance. Chimeric
oligonucleotides having 2'-MOE substituents in the wing nucleosides
and an internal region of deoxy-phosphorothioate nucleotides (also
termed a gapped oligonucleotide or gapmer) have shown effective
reduction in the growth of tumors in animal models at low doses.
2'-MOE substituted oligonucleotides have also shown outstanding
promise as antisense agents in several disease states. One such MOE
substituted oligonucleotide is presently being investigated in
clinical trials for the treatment of CMV retinitis.
[0190] Unless otherwise defined herein, alkyl means
C.sub.1-C.sub.12, C.sub.1-C.sub.8, or C.sub.1-C.sub.6, straight or
(where possible) branched chain aliphatic hydrocarbyl.
[0191] Unless otherwise defined herein, heteroalkyl means
C.sub.1-C.sub.12, C.sub.1-C.sub.8, or C.sub.1-C.sub.6, straight or
(where possible) branched chain aliphatic hydrocarbyl containing at
least one, or about 1 to about 3 hetero atoms in the chain,
including the terminal portion of the chain. Suitable heteroatoms
include N, O and S.
[0192] Unless otherwise defined herein, cycloalkyl means
C.sub.3-C.sub.12, C.sub.3-C.sub.8, or C.sub.3-C.sub.6, aliphatic
hydrocarbyl ring.
[0193] Unless otherwise defined herein, alkenyl means
C.sub.2-C.sub.12, C.sub.2-C.sub.8, or C.sub.2-C.sub.6 alkenyl,
which may be straight or (where possible) branched hydrocarbyl
moiety, which contains at least one carbon-carbon double bond.
[0194] Unless otherwise defined herein, alkynyl means
C.sub.2-C.sub.12, C.sub.2-C.sub.8, or C.sub.2-C.sub.6 alkynyl,
which may be straight or (where possible) branched hydrocarbyl
moiety, which contains at least one carbon-carbon triple bond.
[0195] Unless otherwise defined herein, heterocycloalkyl means a
ring moiety containing at least three ring members, at least one of
which is carbon, and of which 1, 2 or three ring members are other
than carbon. The number of carbon atoms can vary from 1 to about
12, from 1 to about 6, and the total number of ring members varies
from three to about 15, or from about 3 to about 8. Suitable ring
heteroatoms are N, O and S. Suitable heterocycloalkyl groups
include, but are not limited to, morpholino, thiomorpholino,
piperidinyl, piperazinyl, homopiperidinyl, homopiperazinyl,
homomorpholino, homothiomorpholino, pyrrolodinyl,
tetrahydrooxazolyl, tetrahydroimidazolyl, tetrahydrothiazolyl,
tetrahydroisoxazolyl, tetrahydropyrrazolyl, furanyl, pyranyl, and
tetrahydroisothiazolyl.
[0196] Unless otherwise defined herein, aryl means any hydrocarbon
ring structure containing at least one aryl ring. Suitable aryl
rings have about 6 to about 20 ring carbons. Especially suitable
aryl rings include phenyl, napthyl, anthracenyl, and
phenanthrenyl.
[0197] Unless otherwise defined herein, hetaryl means a ring moiety
containing at least one fully unsaturated ring, the ring consisting
of carbon and non-carbon atoms. The ring system can contain about 1
to about 4 rings. The number of carbon atoms can vary from 1 to
about 12, from 1 to about 6, and the total number of ring members
varies from three to about 15, or from about 3 to about 8. Suitable
ring heteroatoms are N, O and S. Suitable hetaryl moieties include,
but are not limited to, pyrazolyl, thiophenyl, pyridyl, imidazolyl,
tetrazolyl, pyridyl, pyrimidinyl, purinyl, quinazolinyl,
quinoxalinyl, benzimidazolyl, benzothiophenyl, etc.
[0198] Unless otherwise defined herein, where a moiety is defined
as a compound moiety, such as hetarylalkyl (hetaryl and alkyl),
aralkyl (aryl and alkyl), etc., each of the sub-moieties is as
defined herein.
[0199] Unless otherwise defined herein, an electron withdrawing
group is a group, such as the cyano or isocyanato group that draws
electronic charge away from the carbon to which it is attached.
Other electron withdrawing groups of note include those whose
electronegativities exceed that of carbon, for example halogen,
nitro, or phenyl substituted in the ortho- or para-position with
one or more cyano, isothiocyanato, nitro or halo groups.
[0200] Unless otherwise defined herein, the terms halogen and halo
have their ordinary meanings. Suitable halo (halogen) substituents
are Cl, Br, and I.
[0201] The aforementioned optional substituents are, unless
otherwise herein defined, suitable substituents depending upon
desired properties. Included are halogens (Cl, Br, I), alkyl,
alkenyl, and alkynyl moieties, NO.sub.2, NH.sub.3 (substituted and
unsubstituted), acid moieties (e.g. --CO.sub.2H,
--OSO.sub.3H.sub.2, etc.), heterocycloalkyl moieties, hetaryl
moieties, aryl moieties, etc.
[0202] In all the preceding formulae, the squiggle (.about.)
indicates a bond to an oxygen or sulfur of the 5'-phosphate.
[0203] Phosphate protecting groups include those described in U.S.
Pat. No. 5,760,209, U.S. Pat. No. 5,614,621, U.S. Pat. No.
6,051,699, U.S. Pat. No. 6,020,475, U.S. Pat. No. 6,326,478, U.S.
Pat. No. 6,169,177, U.S. Pat. No. 6,121,437, U.S. Pat. No.
6,465,628.
[0204] Screening methods for the identification of effective
modulators of small non-coding RNAs are also comprehended by the
instant invention and comprise the steps of contacting a small
non-coding RNA, or portion thereof, with one or more candidate
modulators, and selecting for one or more candidate modulators
which decrease or increase the levels, expression or alter the
function of the small non-coding RNA. Once it is shown that the
candidate modulator or modulators are capable of modulating (e.g.
either decreasing or increasing) the levels, expression or altering
the function of the small non-coding RNA, the modulator may then be
employed in further investigative studies, or for use as a target
validation, research, diagnostic, or therapeutic agent in
accordance with the present invention.
[0205] As one non-limiting example, expression patterns within
cells or tissues treated with one or more oligomeric compounds or
compositions of the invention are compared to control cells or
tissues not treated with the compounds or compositions and the
patterns produced are analyzed for differential levels of nucleic
acid expression as they pertain, for example, to disease
association, signaling pathway, cellular localization, expression
level, size, structure or function of the genes examined. These
analyses can be performed on stimulated or unstimulated cells and
in the presence or absence of other compounds that affect
expression patterns.
[0206] The effects of oligomeric compounds on target nucleic acid
expression or function can be tested in any of a variety of cell
types provided that the target nucleic acid is present at
measurable levels. This can be readily determined by methods
routine in the art, for example Northern blot analysis,
ribonuclease protection assays, or real-time RT-PCR. The following
cell types are provided for illustrative purposes, but other cell
types can be routinely used, provided that the target is present in
the cell type chosen.
[0207] T-24 cells: The human transitional cell bladder carcinoma
cell line T-24 is obtained from the American Type Culture
Collection (ATCC) (Manassas, Va.). T-24 cells were routinely
cultured in complete McCoy's 5A basal media (Invitrogen
Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf
serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100
units/mL, and streptomycin 100 .mu.g/mL (Invitrogen Corporation,
Carlsbad, Calif.). Cells were routinely passaged by trypsinization
and dilution when they reached 90% confluence. For Northern
blotting or other analyses, cells harvested when they reached 90%
confluence. Cells were seeded into 96-well plates (Falcon-Primaria
#353872) at a density of 7000 cells/well for use in real-time
RT-PCR analysis.
[0208] A549 cells: The human lung carcinoma cell line A549 is
obtained from the American Type Culture Collection (ATCC)
(Manassas, Va.). A549 cells were routinely cultured in DMEM basal
media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with
10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.),
penicillin 100 units/mL, and streptomycin 100 .mu.g/mL (Invitrogen
Corporation, Carlsbad, Calif.). Cells were routinely passaged by
trypsinization and dilution when they reached 90% confluence.
[0209] HMECs: Normal human mammary epithelial cells (HMECs) are
obtained from American Type Culture Collection (Manassus, Va.).
HMECs are routinely cultured in DMEM high glucose (Invitrogen Life
Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine
serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are
routinely passaged by trypsinization and dilution when they reach
approximately 90% confluence. HMECs are plated in 24-well plates
(Falcon-Primaria # 353047, BD Biosciences, Bedford, MA) at a
density of 50,000-60,000 cells per well, and allowed to attach
overnight prior to treatment with oligomeric compounds. HMECs are
plated in 96-well plates (Falcon-Primaria #353872, BD Biosciences,
Bedford, Mass.) at a density of approximately 10,000 cells per well
and allowed to attach overnight prior to treatment with oligomeric
compounds.
[0210] MCF7 cells: The breast carcinoma cell line MCF7 is obtained
from American Type Culture Collection (Manassus, Va.). MCF7 cells
are routinely cultured in DMFM high glucose (Invitrogen Life
Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine
serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are
routinely passaged by trypsinization and dilution when they reach
approximately 90% confluence. MCF7 cells are plated in 24-well
plates (Falcon-Primaria # 353047, BD Biosciences, Bedford, Mass.)
at a density of approximately 140,000 cells per well, and allowed
to attach overnight prior to treatment with oligomeric compounds.
MCF7 cells are plated in 96-well plates (Falcon-Primaria #353872,
BD Biosciences, Bedford, Mass.) at a density of approximately
20,000 cells per well and allowed to attach overnight prior to
treatment with oligomeric compounds.
[0211] T47D cells: The breast carcinoma cell line T47D is obtained
from American Type Culture Collection (Manassus, Va.). T47D cells
are deficient in expression of the tumor suppressor gene p53. T47D
cells are cultured in DMEM high glucose (Invitrogen Life
Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine
serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are
routinely passaged by trypsinization and dilution when they reach
approximately 90% confluence. T47D cells are plated in 24-well
plates (Falcon-Primaria # 353047, BD Biosciences, Bedford, Mass.)
at a density of approximately 170,000 cells per well, and allowed
to attach overnight prior to treatment with oligomeric compounds.
T47D cells are plated in 96-well plates (Falcon-Primaria #353872,
BD Biosciences, Bedford, Mass.) at a density of approximately
20,000 cells per well and allowed to attach overnight prior to
treatment with oligomeric compounds.
[0212] BJ cells: The normal human foreskin fibroblast BJ cell line
was obtained from American Type Culture Collection (Manassus, Va.).
BJ cells were routinely cultured in MEM high glucose with 2 mM
L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium
bicarbonate and supplemented with 10% fetal bovine serum, 0.1 mM
non-essential amino acids and 1.0 mM sodium pyruvate (all media and
supplements from Invitrogen Life Technologies, Carlsbad, Calif.).
Cells were routinely passaged by trypsinization and dilution when
they reached approximately 80% confluence. Cells were plated on
collagen-coated 24-well plates (Falcon-Primaria #3047, BD
Biosciences, Bedford, Mass.) at approximately 50,000 cells per
well, and allowed to attach to wells overnight.
[0213] B16-F10 cells: The mouse melanoma cell line B16-F10 was
obtained from American Type Culture Collection (Manassas, Va.).
B16-F10 cells were routinely cultured in DMEM high glucose
(Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with
10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad,
Calif.). Cells were routinely passaged by trypsinization and
dilution when they reached approximately 80% confluence. Cells were
seeded into collagen-coated 24-well plates (Falcon-Primaria #3047,
BD Biosciences, Bedford, Mass.) at approximately 50,000 cells per
well and allowed to attach overnight.
[0214] HUVECs: Human vascular endothelial cells (HUVECs) are
obtained from American Type Culture Collection (Manassus, Va.).
HUVECs are routinely cultured in EBM (Clonetics Corporation,
Walkersville, Md.) supplemented with SingleQuots supplements
(Clonetics Corporation, Walkersville, Md.). Cells are routinely
passaged by trypsinization and dilution when they reach
approximately 90% confluence and are maintained for up to 15
passages. HUVECs are plated at approximately 3000 cells/well in
96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford,
Mass.) and treated with oligomeric compounds one day later.
[0215] NHDF cells: Human neonatal dermal fibroblast (NHDF) cells
are obtained from the Clonetics Corporation (Walkersville, Md.).
NHDFs were routinely maintained in Fibroblast Growth Medium
(Clonetics Corporation, Walkersville, Md.) supplemented as
recommended by the supplier. Cells were maintained for up to 10
passages as recommended by the supplier.
[0216] HEK cells: Human embryonic keratinocytes (HEK) are obtained
from the Clonetics Corporation (Walkersville, Md.). HEKs were
routinely maintained in Keratinocyte Growth Medium (Clonetics
Corporation, Walkersville, Md.) formulated as recommended by the
supplier. Cells were routinely maintained for up to 10 passages as
recommended by the supplier.
[0217] 293T cells: The human 293T cell line is obtained from
American Type Culture Collection (Manassas, Va.). 293T cells are a
highly transfectable cell line constitutively expressing the simian
virus 40 (SV40) large T antigen. 293T cells were maintained in
Dulbeccos' Modified Medium (DMEM) (Invitrogen Corporation,
Carlsbad, Calif.) supplemented with 10% fetal calf serum and
antibiotics (Life Technologies).
[0218] HepG2 cells: The human hepatoblastoma cell line HepG2 is
obtained from the American Type Culture Collection (ATCC)
(Manassas, Va.). HepG2 cells are routinely cultured in Eagle's MEM
supplemented with 10% fetal bovine serum, 1 mM non-essential amino
acids, and 1 mM sodium pyruvate (medium and all supplements from
Invitrogen Life Technologies, Carlsbad, Calif.). Cells are
routinely passaged by trypsinization and dilution when they reach
approximately 90% confluence. For treatment with oligomeric
compounds, cells are seeded into 96-well plates (Falcon-Primaria
#353872, BD Biosciences, Bedford, Mass.) at a density of
approximately 7000 cells/well prior to treatment with oligomeric
compounds. For the caspase assay, cells are seeded into collagen
coated 96-well plates (BIOCOAT cellware, Collagen type I, B-D
#354407/356,407, Becton Dickinson, Bedford, Mass.) at a density of
7500 cells/well.
[0219] Preadipocytes: Human preadipocytes are obtained from
Zen-Bio, Inc. (Research Triangle Park, N.C.). Preadipocytes were
routinely maintained in Preadipocyte Medium (ZenBio, Inc., Research
Triangle Park, N.C.) supplemented with antibiotics as recommended
by the supplier. Cells were routinely passaged by trypsinization
and dilution when they reached 90% confluence. Cells were routinely
maintained for up to 5 passages as recommended by the supplier. To
induce differentiation of preadipocytes, cells are then incubated
with differentiation media consisting of Preadipocyte Medium
further supplemented with 2% more fetal bovine serum (final total
of 12%), amino acids, 100 nM insulin, 0.5 mM IBMX, 1 .mu.M
dexamethasone and 1 .mu.M BRL49653. Cells are left in
differentiation media for 3-5 days and then re-fed with adipocyte
media consisting of Preadipocyte Medium supplemented with 33 .mu.M
biotin, 17 .mu.M pantothenate, 100 nM insulin and 1 .mu.M
dexamethasone. Cells differentiate within one week. At this point
cells are ready for treatment with the oligomeric compounds of the
invention. One day prior to transfection, 96-well plates
(Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) are
seeded with approximately 3000 cells/well prior to treatment with
oligomeric compounds.
[0220] Differentiated adipocytes: Human adipocytes are obtained
from Zen-Bio, Inc. (Research Triangle Park, N.C.). Adipocytes were
routinely maintained in Adipocyte Medium (ZenBio, Inc., Research
Triangle Park, N.C.) supplemented with antibiotics as recommended
by the supplier. Cells were routinely passaged by trypsinization
and dilution when they reached 90% confluence. Cells were routinely
maintained for up to 5 passages as recommended by the supplier.
[0221] NT2 cells: The NT2 cell line is obtained from the American
Type Culture Collection (ATCC; Manassa, Va.). The NT2 cell line,
which has the ATCC designation NTERA-2 c1.D1, is a pluripotent
human testicular embryonal carcinoma cell line derived by cloning
the NTERA-2 cell line. The parental NTERA-2 line was established in
1980 from a nude mouse xenograft of the Tera-2 cell line (ATCC
HTB-106). NT2 cells were routinely cultured in DMEM, high glucose
(Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%
fetal bovine serum (Invitrogen Corporation, Carlsbad, Calif.).
Cells were routinely passaged by trypsinization and dilution when
they reached 90% confluence. For Northern blotting or other
analyses, cells harvested when they reached 90% confluence.
[0222] HeLa cells: The human epitheloid carcinoma cell line HeLa is
obtained from the American Tissue Type Culture Collection
(Manassas, Va.). HeLa cells were routinely cultured in DMEM, high
glucose (Invitrogen Corporation, Carlsbad, Calif.) supplemented
with 10% fetal bovine serum (Invitrogen Corporation, Carlsbad,
Calif.). Cells were routinely passaged by trypsinization and
dilution when they reached 90% confluence. For Northern blotting or
other analyses, cells were harvested when they reached 90%
confluence.
[0223] For Northern blotting or other analysis, cells may be seeded
onto 100 mm or other standard tissue culture plates and treated
similarly, using appropriate volumes of medium and
oligonucleotide.
[0224] In general, when cells reach approximately 80% confluency,
they are treated with oligomeric compounds of the invention.
Oligomeric compounds are introduced into cells using the cationic
lipid transfection reagent LIPOFECTIN.TM. (Invitrogen Life
Technologies, Carlsbad, Calif.). Oligomeric compounds are mixed
with LIPOFECTIN.TM. in OPTI-MEM.TM. (Invitrogen Life Technologies,
Carlsbad, Calif.) to achieve the desired final concentration of
oligomeric compound and LIPOFECTIN.TM.. Before adding to cells, the
oligomeric compound, LIPOFECTIN.TM. and OPTI-MEM.TM. are mixed
thoroughly and incubated for approximately 0.5 hrs. The medium is
removed from the plates and the plates are tapped on sterile gauze.
Each well of a 96-well plate is washed with 150 .mu.l of
phosphate-buffered saline or Hank's balanced salt solution. Each
well of a 24-well plate is washed with 250 .mu.L of
phosphate-buffered saline or Hank's balanced salt solution. The
wash buffer in each well is replaced with 100 .mu.L or 250 .mu.L of
the oligomeric compound/OPTI-MEM.TM./LIPOFECTIN.TM. cocktail for
96-well or 24-well plates, respectively. Untreated control cells
receive LIPOFECTIN.TM. only. The plates are incubated for
approximately 4 to 7 hours at 37.degree. C., after which the medium
is removed and the plates are tapped on sterile gauze. 100 .mu.l or
1 mL of full growth medium is added to each well of a 96-well plate
or a 24-well plate, respectively. Cells are harvested 16-24 hours
after oligonucleotide treatment, at which time RNA can be isolated
and target reduction measured by real-time RT-PCR, or other
phenotypic assays performed. In general, data from treated cells
are obtained in triplicate, and results presented as an average of
the three trials.
[0225] In some embodiments, cells are transiently transfected with
oligomeric compounds of the instant invention. In some embodiments,
cells are transfected and selected for stable expression of an
oligomeric compound of the instant invention.
[0226] Examples of methods of gene expression analysis known in the
art include DNA arrays or microarrays (Brazma et al., FEBS Lett.,
2000, 480, 17-24; Celis et al., FEBS Lett., 2000, 480, 2-16), SAGE
(serial analysis of gene expression)(Madden et al., Drug Discov.
Today, 2000, 5, 415-425), READS (restriction enzyme amplification
of digested cDNAs) (Prashar et al., Methods Enzymol., 1999, 303,
258-72), TOGA (total gene expression analysis) (Sutcliffe et al.,
Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays
and proteomics (Celis et al., FEBS Lett., 2000, 480, 2-16; Jungblut
et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag
(EST) sequencing (Celis et al., FEBS Lett., 2000, 480, 2-16;
Larsson et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA
fingerprinting (SuRF) (Fuchs et al., Anal. Biochem., 2000, 286,
91-98; Larson et al., Cytometry, 2000, 41, 203-208), subtractive
cloning, differential display (DD) (Jurecic et al., Curr. Opin.
Microbiol., 2000, 3, 316-21), comparative genomic hybridization
(Carulli et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH
(fluorescent in situ hybridization) techniques (Going et al., Eur.
J. Cancer, 1999, 35, 1895-904), mass spectrometry methods (To,
Comb. Chem. High Throughput Screen, 2000, 3, 235-41) and real-time
quantitative RT-PCR (Heid et al., Genome Res., 1996, 6,
986-94).
[0227] Modulation of target levels or expression can be assayed in
a variety of ways known in the art. For example, target nucleic
acid levels can be quantitated by, e.g., Northern blot analysis,
competitive polymerase chain reaction (PCR), or real-time
quantitative RT-PCR (also known as RT-PCR). Real-time quantitative
RT-PCR is presently preferred. RNA analysis can be performed on
total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are
well known in the art. Northern blot analysis is also routine in
the art. Real-time quantitative RT-PCR can be conveniently
accomplished using the commercially available ABI PRISM.TM. 7600,
7700, or 7900 Sequence Detection System, available from PE-Applied
Biosystems, Foster City, Calif. and used according to
manufacturer's instructions.
[0228] Poly(A)+ mRNA isolation: Poly(A)+ mRNA was isolated
according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764).
Other methods for poly(A)+ mRNA isolation are routine in the art.
Briefly, for cells grown on 96-well plates, growth medium was
removed from the cells and each well was washed with 200 .mu.L cold
phosphate-buffered saline (PBS). 60 .mu.L lysis buffer (10 mM
Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM
vanadyl-ribonucleoside complex) was added to each well, the plate
was gently agitated and then incubated at room temperature for five
minutes. 55 .mu.L of lysate was transferred to Oligo d(T) coated
96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated
for 60 minutes at room temperature, washed 3 times with 200 .mu.L
of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl).
After the final wash, the plate was blotted on paper towels to
remove excess wash buffer and then air-dried for 5 minutes. 60
.mu.L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to
70.degree. C., was added to each well, the plate was incubated on a
90.degree. C. hot plate for 5 minutes, and the eluate was then
transferred to a fresh 96-well plate.
[0229] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
[0230] Total RNA Isolation: Total RNA was isolated using an RNEASY
96.TM. kit and buffers purchased from Qiagen Inc. (Valencia,
Calif.) following the manufacturer's recommended procedures.
Briefly, for cells grown on 96-well plates, growth medium was
removed from the cells and each well was washed with 200 .mu.L cold
PBS. 150 .mu.L Buffer RLT was added to each well and the plate
vigorously agitated for 20 seconds. 150 .mu.L of 70% ethanol was
then added to each well and the contents mixed by pipetting three
times up and down. The samples were then transferred to the RNEASY
96.TM. well plate attached to a QIAVAC.TM. manifold fitted with a
waste collection tray and attached to a vacuum source. Vacuum was
applied for 1 minute. 500 .mu.L of Buffer RW1 was added to each
well of the RNEASY 96.TM. plate and incubated for 15 minutes and
the vacuum was again applied for 1 minute. An additional 500 .mu.L
of Buffer RW1 was added to each well of the RNEASY 96.TM. plate and
the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then
added to each well of the RNEASY 96.TM. plate and the vacuum
applied for a period of 90 seconds. The Buffer RPE wash was then
repeated and the vacuum was applied for an additional 3 minutes.
The plate was then removed from the QIAVAC.TM. manifold and blotted
dry on paper towels. The plate was then re-attached to the
QIAVAC.TM. manifold fitted with a collection tube rack containing
1.2 mL collection tubes. RNA was then eluted by pipetting 140 .mu.L
of RNAse free water into each well, incubating 1 minute, and then
applying the vacuum for 3 minutes.
[0231] The repetitive pipetting and elution steps may be automated
using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.).
Essentially, after lysing of the cells on the culture plate, the
plate is transferred to the robot deck where the pipetting, DNase
treatment and elution steps are carried out.
[0232] Real-time Quantitative PCR Analysis of a target RNA Levels:
Quantitation of a target RNA levels was accomplished by real-time
quantitative PCR using the ABI PRISM.TM. 7600, 7700, or 7900
Sequence Detection System (PE-Applied Biosystems, Foster City,
Calif.) according to manufacturer's instructions. This is a
closed-tube, non-gel-based, fluorescence detection system which
allows high-throughput quantitation of polymerase chain reaction
(PCR) products in real-time. As opposed to standard PCR in which
amplification products are quantitated after the PCR is completed,
products in real-time quantitative PCR are quantitated as they
accumulate. This is accomplished by including in the PCR reaction
an oligonucleotide probe that anneals specifically between the
forward and reverse PCR primers, and contains two fluorescent dyes.
A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied
Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda,
CA or Integrated DNA Technologies Inc., Coralville, Iowa) is
attached to the 5' end of the probe and a quencher dye (e.g.,
TAMRA, obtained from either PE-Applied Biosystems, Foster City,
Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA
Technologies Inc., Coralville, Iowa) is attached to the 3' end of
the probe. When the probe and dyes are intact, reporter dye
emission is quenched by the proximity of the 3' quencher dye.
During amplification, annealing of the probe to the target sequence
creates a substrate that can be cleaved by the 5'-exonuclease
activity of Taq polymerase. During the extension phase of the PCR
amplification cycle, cleavage of the probe by Taq polymerase
releases the reporter dye from the remainder of the probe (and
hence from the quencher moiety) and a sequence-specific fluorescent
signal is generated. With each cycle, additional reporter dye
molecules are cleaved from their respective probes, and the
fluorescence intensity is monitored at regular intervals by laser
optics built into the ABI PRISM.TM. Sequence Detection System. In
each assay, a series of parallel reactions containing serial
dilutions of RNA from untreated control samples generates a
standard curve that is used to quantitate the percent inhibition
after oligonucleotide treatment of test samples.
[0233] Prior to quantitative PCR analysis, primer/probe sets
specific to the target gene (or RNA) being measured are evaluated
for their ability to be "multiplexed" with a GAPDH amplification
reaction. In multiplexing, both the target gene (or RNA) and the
internal standard gene GAPDH are amplified concurrently in a single
sample. In this analysis, RNA isolated from untreated cells is
serially diluted. Each dilution is amplified in the presence of
primer/probe sets specific for GAPDH only, target gene (or RNA)
only ("single-plexing"), or both (multiplexing). Following PCR
amplification, standard curves of GAPDH and target RNA signal as a
function of dilution are generated from both the single-plexed and
multiplexed samples. If both the slope and correlation coefficient
of the GAPDH and target signals generated from the multiplexed
samples fall within 10% of their corresponding values generated
from the single-plexed samples, the primer/probe set specific for
that target is deemed multiplexable. Other methods of PCR are also
known in the art.
[0234] PCR reagents were obtained from Invitrogen Corporation,
(Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20
.mu.L PCR cocktail (2.5.times.PCR buffer minus MgCl.sub.2, 6.6 mM
MgCl.sub.2, 375 .mu.M each of dATP, dCTP, dCTP and dGTP, 375 nM
each of forward primer and reverse primer, 125 nM of probe, 4 Units
RNAse inhibitor, 1.25 Units PLATINUM.RTM. Taq, 5 Units MuLV reverse
transcriptase, and 2.5.times.ROX dye) to 96-well plates containing
30 .mu.L total RNA solution (20-200 ng). The RT reaction was
carried out by incubation for 30 minutes at 48.degree. C. Following
a 10 minute incubation at 95.degree. C. to activate the
PLATINUM.RTM. Taq, 40 cycles of a two-step PCR protocol were
carried out: 95.degree. C. for 15 seconds (denaturation) followed
by 60.degree. C. for 1.5 minutes (annealing/extension).
[0235] Gene (or RNA) target quantities obtained by real time RT-PCR
are normalized using either the expression level of GAPDH, a gene
whose expression is constant, or by quantifying total RNA using
RiboGreen.TM. (Molecular Probes, Inc. Eugene, Oreg.). GAPDH
expression is quantified by real time RT-PCR, by being run
simultaneously with the target, multiplexing, or separately. Total
RNA is quantified using RiboGreen.TM. RNA quantification reagent
(Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA
quantification by RiboGreen.TM. are taught in Jones, L. J., et al,
(Analytical Biochemistry, 1998, 265, 368-374).
[0236] In this assay, 170 .mu.L of RiboGreen.TM. working reagent
(RiboGreen.TM. reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA,
pH 7.5) is pipetted into a 96-well plate containing 30 .mu.L
purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE
Applied Biosystems) with excitation at 485 nm and emission at 530
nm.
[0237] Probes and primers are designed to hybridize to the target
sequence.
[0238] Northern blot analysis of target RNA levels: Eighteen hours
after treatment, cell monolayers were washed twice with cold PBS
and lysed in 1 mL RNAZOL.TM. (TEL-TEST "B" Inc., Friendswood,
Tex.). Total RNA was prepared following manufacturer's recommended
protocols. Twenty .mu.g of total RNA was fractionated by
electrophoresis through 1.2% agarose gels containing 1.1%
formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon,
Ohio). RNA was transferred from the gel to HYBOND.TM.-N+nylon
membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by
overnight capillary transfer using a Northern/Southern Transfer
buffer system (TEL-TEST "B" Inc., Friendswood, Tex.). RNA transfer
was confirmed by UV visualization. Membranes were fixed by UV
cross-linking using a STRATALINKER.TM. UV Crosslinker 2400
(Stratagene, Inc, La Jolla, Calif.) and then probed using
QUICKHYB.TM. hybridization solution (Stratagene, La Jolla, Calif.)
using manufacturer's recommendations for stringent conditions.
[0239] To detect a target, a target specific primer/probe set is
prepared for analysis by PCR. To normalize for variations in
loading and transfer efficiency, membranes can be stripped and
probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
RNA (Clontech, Palo Alto, Calif.).
[0240] Hybridized membranes were visualized and quantitated using a
PHOSPHORIMAGER.TM. and IMAGEQUANT.TM. Software V3.3 (Molecular
Dynamics, Sunnyvale, Calif.). Data can be normalized to GAPDH
levels in untreated controls.
[0241] The compounds and compositions of the invention are useful
for research and diagnostics, because these compounds and
compositions hybridize to nucleic acids or interfere with the
normal function of these nucleic acids. Hybridization of the
compounds and compositions of the invention with a nucleic acid can
be detected by means known in the art. Such means may include
conjugation of an enzyme to the compound or composition,
radiolabeling or any other suitable detection means. Kits using
such detection means for detecting the level of selected proteins
in a sample may also be prepared.
[0242] The specificity and sensitivity of compounds and
compositions can also be harnessed by those of skill in the art for
therapeutic uses. Antisense oligomeric compounds have been employed
as therapeutic moieties in the treatment of disease states in
animals, including humans. Antisense oligonucleotide drugs,
including ribozymes, have been safely and effectively administered
to humans and numerous clinical trials are presently underway. It
is thus established that oligomeric compounds can be useful
therapeutic modalities that can be configured to be useful in
treatment regimes for the treatment of cells, tissues and animals,
especially humans.
[0243] For therapeutics, an animal, preferably a human, suspected
of having a disease or disorder presenting conditions that can be
treated, ameliorated, or improved by modulating the expression of a
selected small non-coding target nucleic acid is treated by
administering the compounds and compositions. For example, in one
non-limiting embodiment, the methods comprise the step of
administering to or contacting the animal, an effective amount of a
modulator or mimic to treat, ameliorate or improve the conditions
associated with the disease or disorder. The compounds of the
present invention effectively modulate the activity or function of
the small non-coding RNA target or inhibit the expression or levels
of the small non-coding RNA target. In one embodiment, the activity
or expression of the target in an animal is inhibited by about 10%.
In another embodiment the activity or expression of a target in an
animal is inhibited by about 30%. Further, the activity or
expression of a target in an animal is inhibited by 50% or more, by
60% or more, by 70% or more, by 80% or more, by 90% or more, or by
95% or more. In another embodiment, the present invention provides
for the use of a compound of the invention in the manufacture of a
medicament for the treatment of any and all conditions disclosed
herein.
[0244] The reduction of target levels may be measured in serum,
adipose tissue, liver or any other body fluid, tissue or organ of
the animal known to contain the small non-coding RNA or its
precursor. Further, the cells contained within the fluids, tissues
or organs being analyzed contain a nucleic acid molecule of a
downstream target regulated or modulated by the small non-coding
RNA target itself.
[0245] The oligomeric compounds and compositions of the invention
can be utilized in pharmaceutical compositions by adding an
effective amount of the compound or composition to a suitable
pharmaceutically acceptable diluent or carrier. Use of the
oligomeric compounds and methods of the invention may also be
useful prophylactically.
[0246] The oligomeric compounds and compositions of the invention
may also be admixed, encapsulated, conjugated or otherwise
associated with other molecules, molecule structures or mixtures of
compounds, as for example, liposomes, receptor-targeted molecules,
oral, rectal, topical or other formulations, for assisting in
uptake, distribution and/or absorption. Representative U.S. patents
that teach the preparation of such uptake, distribution and/or
absorption-assisting formulations include, but are not limited to,
U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127;
5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330;
4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221;
5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854;
5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575;
and 5,595,756.
[0247] The oligomeric compounds and compositions of the invention
encompass any pharmaceutically acceptable salts, esters, or salts
of such esters, or any other compound which, upon administration to
an animal, including a human, is capable of providing (directly or
indirectly) the biologically active metabolite or residue thereof.
Accordingly, for example, the disclosure is also drawn to prodrugs
and pharmaceutically acceptable salts of the oligomeric compounds
of the invention, pharmaceutically acceptable salts of such
prodrugs, and other bioequivalents.
[0248] The term "prodrug" indicates a therapeutic agent that is
prepared in an inactive form that is converted to an active form
(i.e., drug) within the body or cells thereof by the action of
endogenous enzymes or other chemicals and/or conditions. In
particular, prodrug versions of the oligomeric compounds of the
invention can be prepared as SATE ((S-acetyl-2-thioethyl)
phosphate) derivatives according to the methods disclosed in WO
93/24510 or in WO 94/26764 and U.S. Pat. No. 5,770,713. Larger
oligomeric compounds that are processed to supply, as cleavage
products, compounds capable of modulating the function or
expression of small non-coding RNAs or their downstream targets are
also considered prodrugs.
[0249] The term "pharmaceutically acceptable salts" refers to
physiologically and pharmaceutically acceptable salts of the
compounds and compositions of the invention: i.e., salts that
retain the desired biological activity of the parent compound and
do not impart undesired toxicological effects thereto. Suitable
examples include, but are not limited to, sodium and postassium
salts. For oligonucleotides, examples of pharmaceutically
acceptable salts and their uses are further described in U.S. Pat.
No. 6,287,860.
[0250] The present invention also includes pharmaceutical
compositions and formulations that include the oligomeric compounds
and compositions of the invention. The pharmaceutical compositions
of the present invention may be administered in a number of ways
depending upon whether local or systemic treatment is desired and
upon the area to be treated. Administration may be topical
(including ophthalmic and to mucous membranes including vaginal and
rectal delivery), pulmonary, e.g., by inhalation or insufflation of
powders or aerosols, including by nebulizer; intratracheal,
intranasal, epidermal and transdermal), oral or parenteral.
Parenteral administration includes intravenous, intraarterial,
subcutaneous, intraperitoneal or intramuscular injection or
infusion; or intracranial, e.g., intrathecal or intraventricular,
administration. Pharmaceutical compositions and formulations for
topical administration may include transdermal patches, ointments,
lotions, creams, gels, drops, suppositories, sprays, liquids and
powders. Conventional pharmaceutical carriers, aqueous, powder or
oily bases, thickeners and the like may be necessary or desirable.
Coated condoms, gloves and the like may also be useful.
[0251] Oligomeric compounds may be formulated for delivery in vivo
in an acceptable dosage form, e.g. as parenteral or non-parenteral
formulations. Parenteral formulations include intravenous (i.v.),
subcutaneous (s.c.), intraperitoneal (i.p.), intravitreal and
intramuscular (i.m.) formulations, as well as formulations for
delivery via pulmonary inhalation, intranasal administration,
topical administration, etc. Non-parenteral formulations include
formulations for delivery via the alimentary canal, e.g. oral
administration, rectal administration, intrajejunal instillation,
etc. Rectal administration includes administration as an enema or a
suppository. Oral administration includes administration as a
capsule, a gel capsule, a pill, an elixir, etc.
[0252] In some embodiments, an oligomeric compound can be
administered to a subject via an oral route of administration. The
subject may be an animal or a human (man). An animal subject may be
a mammal, such as a mouse, a rat, a dog, a guinea pig, a monkey, a
non-human primate, a cat or a pig. Non-human primates include
monkeys and chimpanzees. A suitable animal subject may be an
experimental animal, such as a mouse, rat, mouse, a rat, a dog, a
monkey, a non-human primate, a cat or a pig.
[0253] In some embodiments, the subject may be a human. In certain
embodiments, the subject may be a human patient. In certain
embodiments, the subject may be in need of modulation of expression
of one or more genes as discussed in more detail herein. In some
particular embodiments, the subject may be in need of inhibition of
expression of one or more genes as discussed in more detail herein.
In particular embodiments, the subject may be in need of
modulation, i.e. inhibition or enhancement, of a nucleic acid
target in order to obtain therapeutic indications discussed in more
detail herein.
[0254] In some embodiments, non-parenteral (e.g. oral) oligomeric
compound formulations according to the present invention result in
enhanced bioavailability of the compound. In this context, the term
"bioavailability" refers to a measurement of that portion of an
administered drug which reaches the circulatory system (e.g. blood,
especially blood plasma) when a particular mode of administration
is used to deliver the drug. Enhanced bioavailability refers to a
particular mode of administration's ability to deliver
oligonucleotide to the peripheral blood plasma of a subject
relative to another mode of administration. For example, when a
non-parenteral mode of administration (e.g. an oral mode) is used
to introduce the drug into a subject, the bioavailability for that
mode of administration may be compared to a different mode of
administration, e.g. an IV mode of administration. In some
embodiments, the area under a compound's blood plasma concentration
curve (AUC.sub.0) after non-parenteral (e.g. oral, rectal,
intrajejunal) administration may be divided by the area under the
drug's plasma concentration curve after intravenous (i.v.)
administration (AUC.sub.iv) to provide a dimensionless quotient
(relative bioavailability, RB) that represents the fraction of
compound absorbed via the non-parenteral route as compared to the
IV route. A composition's bioavailability is said to be enhanced in
comparison to another composition's bioavailability when the first
composition's relative bioavailability (RB.sub.1) is greater than
the second composition's relative bioavailability (RB.sub.2).
[0255] In general, bioavailability correlates with therapeutic
efficacy when a compound's therapeutic efficacy is related to the
blood concentration achieved, even if the drug's ultimate site of
action is intracellular (van Berge-Henegouwen et al.,
Gastroenterol., 1977, 73, 300). Bioavailability studies have been
used to determine the degree of intestinal absorption of a drug by
measuring the change in peripheral blood levels of the drug after
an oral dose (DiSanto, Chapter 76 In: Remington's Pharmaceutical
Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,
1990, pages 1451-1458).
[0256] In general, an oral composition's bioavailability is said to
be "enhanced" when its relative bioavailability is greater than the
bioavailability of a composition substantially consisting of pure
oligonucleotide, i.e. oligonucleotide in the absence of a
penetration enhancer.
[0257] Organ bioavailability refers to the concentration of
compound in an organ. Organ bioavailability may be measured in test
subjects by a number of means, such as by whole-body radiography.
Organ bioavailability may be modified, e.g. enhanced, by one or
more modifications to the oligomeric compound, by use of one or
more carrier compounds or excipients. In general, an increase in
bioavailability will result in an increase in organ
bioavailability.
[0258] Oral oligomeric compound compositions according to the
present invention may comprise one or more "mucosal penetration
enhancers," also known as "absorption enhancers" or simply as
"penetration enhancers." Accordingly, some embodiments of the
invention comprise at least one oligomeric compound in combination
with at least one penetration enhancer. In general, a penetration
enhancer is a substance that facilitates the transport of a drug
across mucous membrane(s) associated with the desired mode of
administration, e.g. intestinal epithelial membranes. Accordingly
it is desirable to select one or more penetration enhancers that
facilitate the uptake of one or more oligomeric compounds, without
interfering with the activity of the compounds, and in such a
manner the compounds can be introduced into the body of an animal
without unacceptable side-effects such as toxicity, irritation or
allergic response.
[0259] Embodiments of the present invention provide compositions
comprising one or more pharmaceutically acceptable penetration
enhancers, and methods of using such compositions, which result in
the improved bioavailability of oligomeric compounds administered
via non-parenteral modes of administration. Heretofore, certain
penetration enhancers have been used to improve the bioavailability
of certain drugs. See Muranishi, Crit. Rev. Ther. Drug Carrier
Systems, 1990, 7, 1 and Lee et al., Crit. Rev. Ther. Drug Carrier
Systems, 1991, 8, 91. It has been found that the uptake and
delivery of oligonucleotides can be greatly improved even when
administered by non-parenteral means through the use of a number of
different classes of penetration enhancers.
[0260] In some embodiments, compositions for non-parenteral
administration include one or more modifications from
naturally-occurring oligonucleotides (i.e. full-phosphodiester
deoxyribosyl or full-phosphodiester ribosyl oligonucleotides). Such
modifications may increase binding affinity, nuclease stability,
cell or tissue permeability, tissue distribution, or other
biological or pharmacokinetic property. Modifications may be made
to the base, the linker, or the sugar, in general, as discussed in
more detail herein with regards to oligonucleotide chemistry. In
some embodiments of the invention, compositions for administration
to a subject, and in particular oral compositions for
administration to an animal or human subject, will comprise
modified oligonucleotides having one or more modifications for
enhancing affinity, stability, tissue distribution, or other
biological property.
[0261] Suitable modified linkers include phosphorothioate linkers.
In some embodiments according to the invention, the oligomeric
compound has at least one phosphorothioate linker. Phosphorothioate
linkers provide nuclease stability as well as plasma protein
binding characteristics to the compound. Nuclease stability is
useful for increasing the in vivo lifetime of oligomeric compounds,
while plasma protein binding decreases the rate of first pass
clearance of oligomeric compound via renal excretion. In some
embodiments according to the present invention, the oligomeric
compound has at least two phosphorothioate linkers. In some
embodiments, wherein the oligomeric compound has exactly n
nucleosides, the oligomeric compound has from one to n-1
phosphorothioate linkages. In some embodiments, wherein the
oligomeric compound has exactly n nucleosides, the oligomeric
compound has n-1 phosphorothioate linkages. In other embodiments
wherein the oligomeric compound has exactly n nucleoside, and n is
even, the oligomeric compound has from 1 to n/2 phosphorothioate
linkages, or, when n is odd, from 1 to (n-1)/2 phosphorothioate
linkages. In some embodiments, the oligomeric compound has
alternating phosphodiester (PO) and phosphorothioate (PS) linkages.
In other embodiments, the oligomeric compound has at least one
stretch of two or more consecutive PO linkages and at least one
stretch of two or more PS linkages. In other embodiments, the
oligomeric compound has at least two stretches of PO linkages
interrupted by at least one PS linkage.
[0262] In some embodiments, at least one of the nucleosides is
modified on the ribosyl sugar unit by a modification that imparts
nuclease stability, binding affinity or some other beneficial
biological property to the sugar. In some cases, the sugar
modification includes a 2'-modification, e.g. the 2'-OH of the
ribosyl sugar is replaced or substituted. Suitable replacements for
2'-OH include 2'-F and 2'-arabino-F. Suitable substitutions for OH
include 2'-O-alkyl, e.g. 2'-O-methyl, and 2'-O-substituted alkyl,
e.g. 2'-O-methoxyethyl, 2'-O-aminopropyl, etc. In some embodiments,
the oligomeric compound contains at least one 2'-modification. In
some embodiments, the oligomeric compound contains at least 2
2'-modifications. In some embodiments, the oligomeric compound has
at least one 2'-modification at each of the termini (i.e. the 3'-
and 5'-terminal nucleosides each have the same or different
2'-modifications). In some embodiments, the oligomeric compound has
at least two sequential 2'-modifications at each end of the
compound. In some embodiments, oligomeric compounds further
comprise at least one deoxynucleoside. In particular embodiments,
oligomeric compounds comprise a stretch of deoxynucleosides such
that the stretch is capable of activating RNase (e.g. RNase H)
cleavage of an RNA to which the oligomeric compound is capable of
hybridizing. In some embodiments, a stretch of deoxynucleosides
capable of activating RNase-mediated cleavage of RNA comprises
about 8 to about 16, e.g. about 8 to about 16 consecutive
deoxynucleosides. In further embodiments, oligomeric compounds are
capable of eliciting cleaveage by dsRNAse enzymes.
[0263] Oral compositions for administration of non-parenteral
oligomeric compounds and compositions of the present invention may
be formulated in various dosage forms such as, but not limited to,
tablets, capsules, liquid syrups, soft gels, suppositories, and
enemas. The term "alimentary delivery" encompasses e.g. oral,
rectal, endoscopic and sublingual/buccal administration. A common
requirement for these modes of administration is absorption over
some portion or all of the alimentary tract and a need for
efficient mucosal penetration of the nucleic acid(s) so
administered.
[0264] Delivery of a drug via the oral mucosa, as in the case of
buccal and sublingual administration, has several desirable
features, including, in many instances, a more rapid rise in plasma
concentration of the drug than via oral delivery (Harvey, Chapter
35 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed.,
Mack Publishing Co., Easton, Pa., 1990, page 711).
[0265] Endoscopy may be used for delivery directly to an interior
portion of the alimentary tract. For example, endoscopic retrograde
cystopancreatography (ERCP) takes advantage of extended gastroscopy
and permits selective access to the biliary tract and the
pancreatic duct (Hirahata et al., Gan To Kagaku Ryoho, 1992, 19(10
Suppl.), 1591). Pharmaceutical compositions, including liposomal
formulations, can be delivered directly into portions of the
alimentary canal, such as, e.g., the duodenum (Somogyi et al.,
Pharm. Res., 1995, 12, 149) or the gastric submucosa (Akamo et al.,
Japanese J. Cancer Res., 1994, 85, 652) via endoscopic means.
Gastric lavage devices (Inoue et al., Artif. Organs, 1997, 21, 28)
and percutaneous endoscopic feeding devices (Pennington et al.,
Ailment Pharmacol. Ther., 1995, 9, 471) can also be used for direct
alimentary delivery of pharmaceutical compositions.
[0266] In some embodiments, oligomeric compound formulations may be
administered through the anus into the rectum or lower intestine.
Rectal suppositories, retention enemas or rectal catheters can be
used for this purpose and may be preferred when patient compliance
might otherwise be difficult to achieve (e.g., in pediatric and
geriatric applications, or when the patient is vomiting or
unconscious). Rectal administration can result in more prompt and
higher blood levels than the oral route. (Harvey, Chapter 35 In:
Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack
Publishing Co., Easton, Pa., 1990, page 711). Because about 50% of
the drug that is absorbed from the rectum will bypass the liver,
administration by this route significantly reduces the potential
for first-pass metabolism (Benet et al., Chapter 1 In: Goodman
& Gilman's The Pharmacological Basis of Therapeutics, 9th Ed.,
Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996).
[0267] Some embodiments of the present invention employ various
penetration enhancers in order to effect transport of oligomeric
compounds and compositions across mucosal and epithelial membranes.
Penetration enhancers may be classified as belonging to one of five
broad categories--surfactants, fatty acids, bile salts, chelating
agents, and non-chelating non-surfactants (Lee et al., Critical
Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92).
Penetration enhancers and their uses are described in U.S. Pat. No.
6,287,860. Accordingly, some embodiments comprise oral oligomeric
compound compositions comprising at least one member of the group
consisting of surfactants, fatty acids, bile salts, chelating
agents, and non-chelating surfactants. Further embodiments comprise
oral oligomeric compound comprising at least one fatty acid, e.g.
capric or lauric acid, or combinations or salts thereof. Other
embodiments comprise methods of enhancing the oral bioavailability
of an oligomeric compound, the method comprising co-administering
the oligomeric compound and at least one penetration enhancer.
[0268] Other excipients that may be added to oral oligomeric
compound compositions include surfactants (or "surface-active
agents"), which are chemical entities which, when dissolved in an
aqueous solution, reduce the surface tension of the solution or the
interfacial tension between the aqueous solution and another
liquid, with the result that absorption of oligomeric compounds
through the alimentary mucosa and other epithelial membranes is
enhanced. In addition to bile salts and fatty acids, surfactants
include, for example, sodium lauryl sulfate,
polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,
1991, page 92); and perfluorohemical emulsions, such as FC-43
(Takahashi et al., J. Pharm. Phamacol., 1988, 40, 252).
[0269] Fatty acids and their derivatives which act as penetration
enhancers and may be used in compositions of the present invention
include, for example, oleic acid, lauric acid, capric acid
(n-decanoic acid), myristic acid, palmitic acid, stearic acid,
linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein
(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic
acid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one,
acylcarnitines, acylcholines and mono- and di-glycerides thereof
and/or physiologically acceptable salts thereof (i.e., oleate,
laurate, caprate, myristate, palmitate, stearate, linoleate, etc.)
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,
1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug
Carrier Systems, 1990, 7, 1; El-Hariri et al., J. Pharm.
Pharmacol., 1992, 44, 651).
[0270] In some embodiments, oligomeric compound compositions for
oral delivery comprise at least two discrete phases, which phases
may comprise particles, capsules, gel-capsules, microspheres, etc.
Each phase may contain one or more oligomeric compounds,
penetration enhancers, surfactants, bioadhesives, effervescent
agents, or other adjuvant, excipient or diluent. In some
embodiments, one phase comprises at least one oligomeric compound
and at least one penetration enhancer. In some embodiments, a first
phase comprises at least one oligomeric compound and at least one
penetration enhancer, while a second phase comprises at least one
penetration enhancer. In some embodiments, a first phase comprises
at least one oligomeric compound and at least one penetration
enhancer, while a second phase comprises at least one penetration
enhancer and substantially no oligomeric compound. In some
embodiments, at least one phase is compounded with at least one
degradation retardant, such as a coating or a matrix, which delays
release of the contents of that phase. In some embodiments, a first
phase comprises at least one oligomeric compound, at least one
penetration enhancer, while a second phase comprises at least one
penetration enhancer and a release-retardant. In particular
embodiments, an oral oligomeric compound comprises a first phase
comprising particles containing an oligomeric compound and a
penetration enhancer, and a second phase comprising particles
coated with a release-retarding agent and containing penetration
enhancer.
[0271] A variety of bile salts also function as penetration
enhancers to facilitate the uptake and bioavailability of drugs.
The physiological roles of bile include the facilitation of
dispersion and absorption of lipids and fat-soluble vitamins
(Brunton, Chapter 38 In: Goodman & Gilman's The Pharmacological
Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill,
New York, N.Y., 1996, pages 934-935). Various natural bile salts,
and their synthetic derivatives, act as penetration enhancers.
Thus, the term "bile salt" includes any of the naturally occurring
components of bile as well as any of their synthetic derivatives.
The bile salts of the invention include, for example, cholic acid
(or its pharmaceutically acceptable sodium salt, sodium cholate),
dehydrocholic acid (sodium dehydrocholate), deoxycholic acid
(sodium deoxycholate), glucholic acid (sodium glucholate),
glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium
glycodeoxycholate), taurocholic acid (sodium taurocholate),
taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic
acid (CDCA, sodium chenodeoxycholate), ursodeoxycholic acid (UDCA),
sodium tauro-24,25-dihydro-fusidate (STDHF), sodium
glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee
et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical
Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,
1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic
Drug Carrier Systems, 1990, 7, 1; Yamamoto et al., J. Pharm. Exp.
Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79,
579).
[0272] In some embodiments, penetration enhancers useful in some
embodiments of present invention are mixtures of penetration
enhancing compounds. One such penetration enhancer is a mixture of
UDCA (and/or CDCA) with capric and/or lauric acids or salts thereof
e.g. sodium. Such mixtures are useful for enhancing the delivery of
biologically active substances across mucosal membranes, in
particular intestinal mucosa. Other penetration enhancer mixtures
comprise about 5-95% of bile acid or salt(s) UDCA and/or CDCA with
5-95% capric and/or lauric acid. Particular penetration enhancers
are mixtures of the sodium salts of UDCA, capric acid and lauric
acid in a ratio of about 1:2:2 respectively. Anther such
penetration enhancer is a mixture of capric and lauric acid (or
salts thereof) in a 0.01:1 to 1:0.01 ratio (mole basis). In
particular embodiments capric acid and lauric acid are present in
molar ratios of, for example, about 0.1:1 to about 1:0.1, in
particular about 0.5:1 to about 1:0.5.
[0273] Other excipients include chelating agents, i.e. compounds
that remove metallic ions from solution by forming complexes
therewith, with the result that absorption of oligomeric compounds
through the alimentary and other mucosa is enhanced. With regard to
their use as penetration enhancers in the present invention,
chelating agents have the added advantage of also serving as DNase
inhibitors, as most characterized DNA nucleases require a divalent
metal ion for catalysis and are thus inhibited by chelating agents
(Jarrett, J. Chromatogr., 1993, 618, 315). Chelating agents of the
invention include, but are not limited to, disodium
ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,
sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl
derivatives of collagen, laureth-9 and N-amino acyl derivatives of
beta-diketones (enamines)(Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi,
Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1;
Buur et al., J. Control Rel., 1990, 14, 43).
[0274] As used herein, non-chelating non-surfactant penetration
enhancers may be defined as compounds that demonstrate
insignificant activity as chelating agents or as surfactants but
that nonetheless enhance absorption of oligomeric compounds through
the alimentary and other mucosal membranes (Muranishi, Critical
Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1). This
class of penetration enhancers includes, but is not limited to,
unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone
derivatives (Lee et al., Critical Reviews in Therapeutic Drug
Carrier Systems, 1991, page 92); and non-steroidal
anti-inflammatory agents such as diclofenac sodium, indomethacin
and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987,
39, 621).
[0275] Agents that enhance uptake of oligomeric compounds at the
cellular level may also be added to the pharmaceutical and other
compositions of the present invention. For example, cationic
lipids, such as lipofectin (U.S. Pat. No. 5,705,188), cationic
glycerol derivatives, and polycationic molecules, such as
polylysine (PCT Application WO 97/30731), can be used.
[0276] Some oral oligomeric compound compositions also incorporate
carrier compounds in the formulation. As used herein, "carrier
compound" or "carrier" can refer to a nucleic acid, or analog
thereof, which may be inert (i.e., does not possess biological
activity per se) or may be necessary for transport, recognition or
pathway activation or mediation, or is recognized as a nucleic acid
by in vivo processes that reduce the bioavailability of an
oligomeric compound having biological activity by, for example,
degrading the biologically active oligomeric compound or promoting
its removal from circulation. The coadministration of a oligomeric
compound and a carrier compound, typically with an excess of the
latter substance, can result in a substantial reduction of the
amount of oligomeric compound recovered in the liver, kidney or
other extracirculatory reservoirs, presumably due to competition
between the carrier compound and the oligomeric compound for a
common receptor. For example, the recovery of a partially
phosphorothioate oligomeric compound in hepatic tissue can be
reduced when it is coadministered with polyinosinic acid, dextran
sulfate, polycytidic acid or
4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et
al., Antisense Res. Dev., 1995, 5, 115; Takakura et al., Antisense
& Nucl. Acid Drug Dev., 1996, 6, 177).
[0277] A "pharmaceutical carrier" or "excipient" may be a
pharmaceutically acceptable solvent, suspending agent or any other
pharmacologically inert vehicle for delivering one or more
oligomeric compounds to an animal. The excipient may be liquid or
solid and is selected, with the planned manner of administration in
mind, so as to provide for the desired bulk, consistency, etc.,
when combined with an oligomeric compound and the other components
of a given pharmaceutical composition. Typical pharmaceutical
carriers include, but are not limited to, binding agents (e.g.,
pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl
methylcellulose, etc.); fillers (e.g., lactose and other sugars,
microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl
cellulose, polyacrylates or calcium hydrogen phosphate, etc.);
lubricants (e.g., magnesium stearate, talc, silica, colloidal
silicon dioxide, stearic acid, metallic stearates, hydrogenated
vegetable oils, corn starch, polyethylene glycols, sodium benzoate,
sodium acetate, etc.); disintegrants (e.g., starch, sodium starch
glycolate, EXPLOTAB); and wetting agents (e.g., sodium lauryl
sulphate, etc.).
[0278] Oral oligomeric compound compositions may additionally
contain other adjunct components conventionally found in
pharmaceutical compositions, at their art-established usage levels.
Thus, for example, the compositions may contain additional,
compatible, pharmaceutically-active materials such as, for example,
antipuritics, astringents, local anesthetics or anti-inflammatory
agents, or may contain additional materials useful in physically
formulating various dosage forms of the composition of present
invention, such as dyes, flavoring agents, preservatives,
antioxidants, opacifiers, thickening agents and stabilizers.
However, such materials, when added, should not unduly interfere
with the biological activities of the components of the
compositions of the present invention.
[0279] The pharmaceutical formulations of the present invention,
which may conveniently be presented in unit dosage form, may be
prepared according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general, the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0280] The oligomeric compounds and compositions of the present
invention may be formulated into any of many possible dosage forms
such as, but not limited to, tablets, capsules, gel capsules,
liquid syrups, soft gels, suppositories, and enemas. The
compositions of the present invention may also be formulated as
suspensions in aqueous, non-aqueous or mixed media. Aqueous
suspensions may further contain substances which increase the
viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
[0281] Pharmaceutical compositions of the present invention
include, but are not limited to, solutions, emulsions, foams and
liposome-containing formulations.
[0282] Emulsions are typically heterogenous systems of one liquid
dispersed in another in the form of droplets usually exceeding 0.1
.mu.m in diameter. Emulsions may contain additional components in
addition to the dispersed phases, and the active drug that may be
present as a solution in either the aqueous phase, oily phase or
itself as a separate phase. Microemulsions are included as an
embodiment of the present invention. Emulsions and their uses are
well known in the art and are described in U.S. Pat. No.
6,287,860.
[0283] Formulations of the present invention include liposomal
formulations. As used in the present invention, the term "liposome"
means a vesicle composed of amphiphilic lipids arranged in a
spherical bilayer or bilayers. Liposomes are unilamellar or
multilamellar vesicles which have a membrane formed from a
lipophilic material and an aqueous interior that contains the
composition to be delivered. Cationic liposomes are positively
charged liposomes which are believed to interact with negatively
charged nucleic acid molecules to form a stable complex. Liposomes
that are pH-sensitive or negatively-charged are believed to entrap
nucleic acids rather than complex with it. Both cationic and
noncationic liposomes have been used to deliver nucleic acids and
oligomeric compounds to cells.
[0284] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids that, when incorporated into liposomes,
result in enhanced circulation lifetimes relative to liposomes
lacking such specialized lipids. Examples of sterically stabilized
liposomes are those in which part of the vesicle-forming lipid
portion of the liposome comprises one or more glycolipids or is
derivatized with one or more hydrophilic polymers, such as a
polyethylene glycol (PEG) moiety. Liposomes and their uses are
described in U.S. Pat. No. 6,287,860.
[0285] The pharmaceutical formulations and compositions of the
present invention may also include surfactants. The use of
surfactants in drug products, formulations and in emulsions is well
known in the art. Surfactants and their uses are described in U.S.
Pat. No. 6,287,860.
[0286] One of skill in the art will recognize that formulations are
routinely designed according to their intended use, i.e. route of
administration.
[0287] Formulations for topical administration include those in
which the oligomeric compounds of the invention are in admixture
with a topical delivery agent such as lipids, liposomes, fatty
acids, fatty acid esters, steroids, chelating agents and
surfactants. Lipids and liposomes include neutral (e.g.
dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl
choline DMPC, distearolyphosphatidyl choline) negative (e.g.
dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.
dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl
ethanolamine DOTMA).
[0288] For topical or other administration, oligomeric compounds
and compositions of the invention may be encapsulated within
liposomes or may form complexes thereto, in particular to cationic
liposomes. Alternatively, they may be complexed to lipids, in
particular to cationic lipids. Topical formulations are described
in detail in U.S. patent application Ser. No. 09/315,298.
[0289] Compositions and formulations for oral administration
include powders or granules, microparticulates, nanoparticulates,
suspensions or solutions in water or non-aqueous media, capsules,
gel capsules, sachets, tablets or minitablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable. Oral formulations are those in which oligomeric
compounds of the invention are administered in conjunction with one
or more penetration enhancers surfactants and chelators. A
particularly suitable combination is the sodium salt of lauric
acid, capric acid and UDCA. Penetration enhancers also include
polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
Compounds and compositions of the invention may be delivered
orally, in granular form including sprayed dried particles, or
complexed to form micro or nanoparticles. Certain oral formulations
for oligonucleotides and their preparation are described in detail
in U.S. application Ser. Nos. 09/108,673 09/315,298, and U.S.
Application Publication 20030027780.
[0290] Compositions and formulations for parenteral, intrathecal or
intraventricular administration may include sterile aqueous
solutions that may also contain buffers, diluents and other
suitable additives such as, but not limited to, penetration
enhancers, carrier compounds and other pharmaceutically acceptable
carriers or excipients.
[0291] Certain embodiments of the invention provide pharmaceutical
compositions containing one or more of the compounds and
compositions of the invention and one or more other
chemotherapeutic agents that function by a non-antisense mechanism.
Examples of such chemotherapeutic agents include but are not
limited to cancer chemotherapeutic drugs such as daunorubicin,
daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin,
esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine
arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,
actinomycin D, mithramycin, prednisone, hydroxyprogesterone,
testosterone, tamoxifen, dacarbazine, procarbazine,
hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine,
chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards,
melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine,
cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,
4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),
5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine,
taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate,
irinotecan, topotecan, gemcitabine, teniposide, cisplatin and
diethylstilbestrol (DES). When used with the oligomeric compounds
of the invention, such chemotherapeutic agents may be used
individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,
5-FU and oligonucleotide for a period of time followed by MTX and
oligonucleotide), or in combination with one or more other such
chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or
5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs,
including but not limited to nonsteroidal anti-inflammatory drugs
and corticosteroids, and antiviral drugs, including but not limited
to ribivirin, vidarabine, acyclovir and ganciclovir, may also be
combined in compositions of the invention. Combinations of
oligomeric compounds and compositions of the invention and other
drugs are also within the scope of this invention. Two or more
combined compounds such as two oligomeric compounds or one
oligomeric compound combined with further compounds may be used
together or sequentially.
[0292] In another embodiment, compositions of the invention may
contain one or more of the compounds and compositions of the
invention targeted to a first nucleic acid target and one or more
additional oligomeric compounds targeted to a second nucleic acid
target. Alternatively, compositions of the invention may contain
two or more oligomeric compounds and compositions targeted to
different regions, segments or sites of the same target. Two or
more combined compounds may be used together or sequentially.
[0293] The formulation of therapeutic compounds and compositions of
the invention and their subsequent administration (dosing) is
believed to be within the skill of those in the art. Dosing is
dependent on severity and responsiveness of the disease state to be
treated, with the course of treatment lasting from several days to
several months, or until a cure is effected or a diminution of the
disease state is achieved. Optimal dosing schedules can be
calculated from measurements of drug accumulation in the body of
the patient. Persons of ordinary skill can easily determine optimum
dosages, dosing methodologies and repetition rates. Optimum dosages
may vary depending on the relative potency of individual oligomeric
compounds, and can generally be estimated based on EC.sub.50s found
to be effective in in vitro and in vivo animal models. In general,
dosage is from 0.01 .mu.g to 100 g per kg of body weight, from 0.1
.mu.g to 10 g per kg of body weight, from 1.0 .mu.g to 1 g per kg
of body weight, from 10.0 .mu.g to 100 mg per kg of body weight,
from 100 .mu.g to 10 mg per kg of body weight, or from 1 mg to 5 mg
per kg of body weight, and may be given once or more daily, weekly,
monthly or yearly, or even once every 2 to 20 years. Persons of
ordinary skill in the art can easily determine repetition rates for
dosing based on measured residence times and concentrations of the
drug in bodily fluids or tissues. Following successful treatment,
it may be desirable to have the patient undergo maintenance therapy
to prevent the recurrence of the disease state, wherein the
oligomeric compound is administered in maintenance doses, ranging
from 0.01 .mu.g to 100 g per kg of body weight, from 0.1 .mu.g to
10 g per kg of body weight, from 1 .mu.g to 1 g per kg of body
weight, from 10 .mu.g to 100 mg per kg of body weight, from 100
.mu.g to 10 mg per kg of body weight, or from 100 .mu.g to 1 mg per
kg of body weight, once or more daily, to once every 20 years. The
effects of treatments with therapeutic compositions can be assessed
following collection of tissues or fluids from a patient or subject
receiving said treatments. It is known in the art that a biopsy
sample can be procured from certain tissues without resulting in
detrimental effects to a patient or subject. In certain
embodiments, a tissue and its constituent cells comprise, but are
not limited to, blood (e.g., hematopoietic cells, such as human
hematopoietic progenitor cells, human hematopoietic stem cells,
CD34.sup.+ cells CD4.sup.+ cells), lymphocytes and other blood
lineage cells, bone marrow, breast, cervix, colon, esophagus, lymph
node, muscle, peripheral blood, oral mucosa and skin. In other
embodiments, a fluid and its constituent cells comprise, but are
not limited to, blood, urine, semen, synovial fluid, lymphatic
fluid and cerebro-spinal fluid. Tissues or fluids procured from
patients can be evaluated for expression levels of a target small
non-coding RNA, mRNA or protein. Additionally, the mRNA or protein
expression levels of other genes known or suspected to be
associated with the specific disease state, condition or phenotype
can be assessed. mRNA levels can be measured or evaluated by
real-time PCR, Northern blot, in situ hybridization or DNA array
analysis.
[0294] The oligomeric compounds of the present invention can also
be formulated into compositions comprising one or more of the
oligomeric compounds described herein. The compositions can contain
an RNA target.
[0295] In order that the invention disclosed herein may be more
efficiently understood, examples are provided below. It should be
understood that these examples are for illustrative purposes only
and are not to be construed as limiting the invention in any
manner. Throughout these examples, molecular cloning reactions, and
other standard recombinant DNA techniques, were carried out
according to methods described in Maniatis et al., Molecular
Cloning--A Laboratory Manual, 2nd ed., Cold Spring Harbor Press
(1989), using commercially available reagents, except where
otherwise noted.
EXAMPLES
Example 1
Sequence Alignment of Pri-mirs
[0296] Fifty human pri-miRNA sequences were analyzed using RNAMOT.
Thirty-eight of the 50 pri-miRNA sequences were confirmed as mir
sequences and 12 were proposed mir sequences. Motifs were
classified by, for example, helical region/length, mismatch pairs,
5' bulged base, and 3' bulged base. In addition, the GC, AU, and GU
content of all helical regions was determined independently.
Sequence alignment yielded no conserved bases near the Drosha
cleavage sites.
[0297] Alignment of the 50 human pri-miRNA sequences resulted in
the following observations for the 5' cleavage site. 19/50 sites
had a 2-base internal mismatch (e.g., UN/CU, where N is U, A, or C;
GN/NG where N is A or U; and CN/CC where N is U or A). Six sites
had 1 base internal mismatch (C/U or A/C). Nineteen sites had a
helical domain. Three sites had an A/C mismatch. Three sites had an
asymmetrical internal bulge.
[0298] Alignment of the 50 human pri-miRNA sequences resulted in
the following observations for the 3' terminus cleavage site. 30/49
sites had a helix of 7 or less base pairs, and all but one had at
least one GU pair. Seven sites had a helix of less than 7 base
pairs, all of which had at least one GU pair. Five sites had A/C
mismatch pairs and five sites had other mismatches (G/A or A/A).
Only two sites had asymmetrical bulges. In general, the helices do
not look very stable (e.g., at least 50% AU with 10-30% GU
pairs).
[0299] Alignment of the 50 human pri-miRNA sequences resulted in
the following observations for the downstream (3') of the 5'
cleavage site. 32/49 sites had a helical domain of 7 base pairs
without a GU pair. Eight sites had a single bulged base in the long
helical domains. Nine sites had A/C or N/N mismatches.
[0300] Alignment of the 50 human pri-miRNA sequences resulted in
the following observations for the downstream of the helical
domain. 44/49 sites had a bulged base, mismatch pairs, or a
destabilizing element.
[0301] Thus, the following conclusions were drawn from these, as
well as other, observations. It would be desirous to have about 8
base pairs of duplex region without a GU pair, separated by a
destabilizing element, such as an A/C mismatch. It would be
desirous to have a UU/UC or GG/AG at the 5' cleavage site. It would
be desirous to have a low stability helical domain at the 3'
cleavage site with at least one GU pair (5'-G, 3'-U) and another
destabilizing element (py/py or A/C mismatch pair). FIGS. 2 and 3
show representative potential motifs to search for in target
mRNAs.
Example 2
Effect of Drosha Sequences on PTEN Expression
[0302] Numerous RNA constructs were prepared for screening in HeLa
cells. HeLa cells were exposed to the RNA constructs at 150 nM for
16 hours. Results are shown in FIGS. 3A, 3B, and 3C.
[0303] Various modifications of the invention, in addition to those
described herein, will be apparent to those skilled in the art from
the foregoing description. Such modifications are also intended to
fall within the scope of the appended claims. Each reference
(including, but not limited to, journal articles, U.S. and non-U.S.
patents, patent application publications, international patent
application publications, gene bank accession numbers, and the
like) cited in the present application is incorporated herein by
reference in its entirety.
Sequence CWU 1
1
54 1 83 RNA H. Sapiens 1 ccuuggagua aaguagcagc acauaauggu
uuguggauuu ugaaaaggug caggccauau 60 ugugcugccu caaaaauaca agg 83 2
98 RNA H. Sapiens 2 uugaggccuu aaaguacugu agcagcacau caugguuuac
augcuacagu caagaugcga 60 aucauuauuu gcugcucuag aaauuuaagg aaauucau
98 3 84 RNA H. Sapiens 3 cuccccaugg cccugucucc caacccuugu
accagugcug ggcucagacc cugguacagg 60 ccugggggac agggaccugg ggac 84 4
87 RNA H. Sapiens 4 uguccccccc ggcccagguu cugugauaca cuccgacucg
ggcucuggag cagucagugc 60 augacagaac uugggcccgg aaggacc 87 5 90 RNA
H. Sapiens 5 cucacagcug ccagugucau uuuugugauc ugcagcuagu auucucacuc
caguugcaua 60 gucacaaaag ugaucauugg cagguguggc 90 6 87 RNA H.
Sapiens 6 agcgguggcc agugucauuu uugugauguu gcagcuagua auaugagccc
aguugcauag 60 ucacaaaagu gaucauugga aacugug 87 7 84 RNA H. Sapiens
7 gugguacuug aagauagguu auccguguug ccuucgcuuu auuugugacg aaucauacac
60 gguugaccua uuuuucagua ccaa 84 8 65 RNA H. Sapiens 8 cuguuaaugc
uaaucgugau agggguuuuu gccuccaacu gacuccuaca uauuagcauu 60 aacag 65
9 71 RNA H. Sapiens 9 guagcacuaa agugcuuaua gugcagguag uguuuaguua
ucuacugcau uaugagcacu 60 uaaaguacug c 71 10 72 RNA H. Sapiens 10
ugucggguag cuuaucagac ugauguugac uguugaaucu cauggcaaca ccagucgaug
60 ggcugucuga ca 72 11 85 RNA H. Sapiens 11 ggcugagccg caguaguucu
ucaguggcaa gcuuuauguc cugacccagc uaaagcugcc 60 aguugaagaa
cuguugcccu cugcc 85 12 73 RNA H. Sapiens 12 ggccggcugg gguuccuggg
gaugggauuu gcuuccuguc acaaaucaca uugccaggga 60 uuuccaaccg acc 73 13
69 RNA H. Sapiens 13 scuccggugc cuacugagcu gauaucaguu cucauuuuac
acacuggcuc aguucagcag 60 gaacaggag 69 14 84 RNA H. Sapiens 14
ggccaguguu gagaggcgga gacuugggca auugcuggac gcugcccugg gcauugcacu
60 ugucucgguc ugacagugcc ggcc 84 15 77 RNA H. Sapiens 15 guggccucgu
ucaaguaauc caggauaggc ugugcagguc ccaaugggcc uauucuuggu 60
uacuugcacg gggacgc 77 16 77 RNA H. Sapiens 16 ccgggaccca guucaaguaa
uucaggauag guugugugcu guccagccug uucuccauua 60 cuuggcucgg ggaccgg
77 17 78 RNA H. Sapiens 17 cugaggagca gggcuuagcu gcuugugagc
aggguccaca ccaagucgug uucacagugg 60 cuaaguuccg ccccccag 78 18 97
RNA H. Sapiens 18 accucucuaa caaggugcag agcuuagcug auuggugaac
agugauuggu uuccgcuuug 60 uucacagugg cuaaguucug caccugaaga gaaggug
97 19 86 RNA H. Sapiens 19 gguccuugcc cucaaggagc ucacagucua
uugaguuacc uuucugacuu ucccacuaga 60 uugugagcuc cuggagggca ggcacu 86
20 64 RNA H. Sapiens 20 augacugauu ucuuuuggug uucagaguca auauaauuuu
cuagcaccau cugaaaucgg 60 uuau 64 21 81 RNA H. Sapiens 21 cuucaggaag
cugguuucau auggugguuu agauuuaaau agugauuguc uagcaccauu 60
ugaaaucagu guucuugggg g 81 22 88 RNA H. Sapiens 22 aucucuuaca
caggcugacc gauuucuccu gguguucaga gucuguuuuu gucuagcacc 60
auuugaaauc gguuaugaug uaggggga 88 23 95 RNA H. Sapiens 23
ccagcucggg cagccguggc caucuuacug ggcagcauug gauggaguca ggucucuaau
60 acugccuggu aaugaugacg gcggagcccu gcacg 95 24 106 RNA H. Sapiens
24 uggggacucg cgcgcugggu ccagugguuc uuaacaguuc aacaguucug
uagcgcaauu 60 gugaaauguu uaggaccacu agacccggcg ggcgcggcga cagcga
106 25 94 RNA H. Sapiens 25 caugugacuc guggacuucc cuuugucauc
cuaugccuga gaauauauga aggaggcugg 60 gaaggcaaag ggacguucaa
uugucaucac uggc 94 26 94 RNA H. Sapiens 26 uccaugugcu ucucuugucc
uucauuccac cggagucugu cucauaccca accagauuuc 60 aguggaguga
aguucaggag gcauggagcu gaca 94 27 86 RNA H. Sapiens 27 ugcuucccga
ggccacaugc uucuuuauau ccccauaugg auuacuuugc uauggaaugu 60
aaggaagugu gugguuucgg caagug 86 28 71 RNA H. Sapiens 28 ugacgggcga
gcuuuuggcc cggguuauac cugaugcuca cguauaagac gagcaaaaag 60
cuuguugguc a 71 29 96 RNA H. Sapiens 29 ccaggcgcag ggcagccccu
gcccaccgca cacugcgcug ccccagaccc acugugcgug 60 ugacagcggc
ugaucugugc cugggcagcg cgaccc 96 30 99 RNA H. Sapiens 30 ugugacuugu
gggcuucccu uugucauccu ucgccuaggg cucugagcag ggcagggaca 60
gcaaaggggu gcucaguugu cacuucccac agcacggag 99 31 95 RNA H. Sapiens
31 ggacagcgcg ccggcaccuu ggcucuagac ugcuuacugc ccgggccgcc
cucaguaaca 60 gucuccaguc acggccaccg acgccuggcc ccgcc 95 32 88 RNA
H. Sapiens 32 accaaguuuc aguucaugua aacauccuac acucagcugu
aauacaugga uuggcuggga 60 gguggauguu uacuucagcu gacuugga 88 33 71
RNA H. Sapiens 33 ggagaggagg caagaugcug gcauagcugu ugaacuggga
accugcuaug ccaacauauu 60 gccaucuuuc c 71 34 70 RNA H. Sapiens 34
ggagauauug cacauuacua aguugcaugu ugucacggcc ucaaugcaau uuagugugug
60 ugauauuuuc 70 35 69 RNA H. Sapiens 35 cuguggugca uuguaguugc
auugcauguu cuggugguac ccaugcaaug uuuccacagu 60 gcaucacag 69 36 84
RNA H. Sapiens 36 gugcucgguu uguaggcagu gucauuagcu gauuguacug
uggugguuac aaucacuaac 60 uccacugcca ucaaaacaag gcac 84 37 86 RNA H.
Sapiens 37 acugcuaacg aaugcucuga cuuuauugca cuacuguacu uuacagcuag
cagugcaaua 60 guauugucaa agcaucugaa agcagg 86 38 101 RNA H. Sapiens
38 ggccuaguuc uguguggaag acuagugauu uuguuguuuu uagauaacua
aaucgacaac 60 aaaucacagu cugccauaug gcacaggcca ugccucuaca g 101 39
89 RNA H. Sapiens 39 cgggguuggu uguuaucuuu gguuaucuag cuguaugagu
gguguggagu cuucauaaag 60 cuagauaacc gaaaguaaaa auaacccca 89 40 78
RNA H. Sapiens 40 cuuucuacac agguugggau cgguugcaau gcuguguuuc
uguaugguau ugcacuuguc 60 ccggccuguu gaguuugg 78 41 80 RNA H.
Sapiens 41 cugggggcuc caaagugcug uucgugcagg uagugugauu acccaaccua
cugcugagcu 60 agcacuuccc gagcccccgg 80 42 81 RNA H. Sapiens 42
aacacagugg gcacucaaua aaugucuguu gaauugaaau gcguuacauu caacggguau
60 uuauugagca cccacucugu g 81 43 78 RNA H. Sapiens 43 uggccgauuu
uggcacuagc acauuuuugc uugugucucu ccgcucugag caaucaugug 60
cagugccaau augggaaa 78 44 81 RNA H. Sapiens 44 cccauuggca
uaaacccgua gauccgaucu uguggugaag uggaccgcac aagcucgcuu 60
cuaugggucu gugucagugu g 81 45 100 RNA H. Sapiens 45 ugucugucuu
cuguauauac ccuguagauc cgaauuugug uaaggaauuu uguggucaca 60
aauucguauc uaggggaaua uguaguugac auaaacacuc 100 46 80 RNA H.
Sapiens 46 ccuguugcca caaacccgua gauccgaacu ugugguauua guccgcacaa
gcuuguaucu 60 auagguaugu gucuguuagg 80 47 78 RNA H. Sapiens 47
uacugcccuc ggcuucuuua cagugcugcc uuguugcaua uggaucaagc agcauuguac
60 agggcuauga aggcauug 78 48 81 RNA H. Sapiens 48 ugugcaucgu
ggucaaaugc ucagacuccu gugguggcug cucaugcacc acggauguuu 60
gagcaugugc uacggugucu a 81 49 82 RNA H. Sapiens 49 ccugccgggg
cuaaagugcu gacagugcag auaguggucc ucuccgugcu accgcacugu 60
ggguacuugc ugcuccagca gg 82 50 80 RNA H. Sapiens 50 ugggaugagg
uaguagguug uauaguuuua gggucacacc caccacuggg agauaacuau 60
acaaucuacu gucuuuccua 80 51 29 RNA Artificial Sequence Sequence
Motif 51 nnnhhhchhh hhaghhhhhh hahhnnnnn 29 52 21 RNA Artificial
Sequence Antisense Compound 52 hhhchhhhha ghhhhhhhah h 21 53 29 RNA
Artificial Sequence Sequence Motif 53 nnnhhhgahh hhuuhhhhhh
hahhnnnnn 29 54 21 RNA Artificial Sequence Antisense Compound 54
hhhgghhhhu chhhhhhhch h 21
* * * * *